CN107090441A - The method based on MGMT for obtaining high yield expression of recombinant proteins - Google Patents
The method based on MGMT for obtaining high yield expression of recombinant proteins Download PDFInfo
- Publication number
- CN107090441A CN107090441A CN201710166781.5A CN201710166781A CN107090441A CN 107090441 A CN107090441 A CN 107090441A CN 201710166781 A CN201710166781 A CN 201710166781A CN 107090441 A CN107090441 A CN 107090441A
- Authority
- CN
- China
- Prior art keywords
- gly
- leu
- ala
- cell
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y201/00—Transferases transferring one-carbon groups (2.1)
- C12Y201/01—Methyltransferases (2.1.1)
- C12Y201/01063—Methylated-DNA-[protein]-cysteine S-methyltransferase (2.1.1.63), i.e. O6-methylguanine-DNA methyltransferase
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/055—Fusion polypeptide containing a localisation/targetting motif containing a signal for localisation to secretory granules (for exocytosis)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
- C12N2015/8518—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/103—Plasmid DNA for invertebrates
- C12N2800/105—Plasmid DNA for invertebrates for insects
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to the method based on MGMT for obtaining high yield expression of recombinant proteins.The invention discloses a kind of novel enhanced of protein production in host cell.It discloses a kind of carrier for being used to express recombinant protein in these cells, peptide signal, b) 6 methyl guanine DNA transmethylases (MGMT, EC 2.1.1.63) and its mutant or catalyst structure domain and c) nucleotide sequence of recombinant protein a) are secreted including coding.The MGMT enzymes are preferably so-called SNAP albumen.
Description
The application is application number 201180065989.9, and the applying date is on December 9th, 2011, entitled " to be used to obtain
High yield expression of recombinant proteins the method based on MGMT " Chinese patent application divisional application.
Technical field
The present invention relates to genetic engineering and biology field.Particularly, the present invention relates to egg in a kind of host cell
Novel enhanced produced in vain.In addition, the present invention relates to the carrier containing the DNA sequence dna for encoding the enhancer albumen, and its
Purposes for expressing recombinant protein, such as industrial enzymes or pharmaceutical protein, including eucaryon (for example, mammal, such as people) and disease
Toxalbumin.
Background technology
Protein production systems, are commercial biological skills wherein producing many peptide or proteins of interest in recombinant organisms or cell
The trunk of art.
Earliest system, based on the bacterial expression in host such as Escherichia coli, has added and is based on eucaryon host
System, the mammalian cell particularly cultivated, culture and whole insect form insect cell, and transgene mammal
Such as sheep and goat.
Prokaryotic culture systems are easy to maintain and operation is cheap.However, prokaryotic can not carry out turning over for eukaryotic protein
Modified after translating.In addition, many albumen can not correctly be folded, it is necessary to which special program makes it fold again, which increase be produced into
This.
A variety of applications have been described in eukaryotic cell culture system.For example, mammalian cell is repaiied after being translated
Decorations, and generally produce correctly fold and soluble protein.The major defect of mammalian cell system includes needing specially
With expensive culture facility, the risk of infection, this may result in the loss of whole culture and is potentially harmful to lactation and moves
The risk of thing protein contamination final products.Insect cell is used alternatingly to be used for expressing polypeptide.It is most universal used in insect cell
Expression system be based on baculovirus vector.Under the control of strong natural polyhedrin promoter, replaced by using heterologous gene
The baculovirus polyhedrin gene of coding baculoviral major structural protein is changed to build rhabdovirus expression vector.With weight
The insect host cell of group virus infection culture, can reclaim resulting albumen from cell is middle in itself, or if using
During suitable secretion signal, resulting albumen is reclaimed from culture medium.
However, both systems, all there is the repeatability of expression of recombinant proteins level and quality, that culture infects is related
Problem, and need special culture facility.In addition, the bar prepared under gmp conditions of having to for some protein productions
Shape viral stocks, not always stablize over time.
Drosophila cell, particularly Drosophila melanogaster (Drosophila melanogaster) S2 cells, by US 5,550,
043rd, it is used for the expression of albumen disclosed in US 5,681,713 and US 5,705,359.Different from the baculoviral system of prior art
System, could provide protein of interest, the method based on S2 cells provides continuous during the insect cell that only cracking is infected wherein
The cell expression system of heterologous protein, and therefore produce higher expression.
Have been proposed several other for strengthening the device that heterologous protein is expressed in host cell：For example, US 5,
919,682 describe in prokaryotes using pCW carriers under the control of tac promoters, coexpression albumen and molecular chaperones
The method of excessive production feature nitric acid synthase.In addition, US4,758,512 are related in its DNA sequence dna with specific mutation
The production of host cell, its ability for causing organism surface to reveal degraded foreign product is reduced.The host organisms of these mutation
Yield available for the foreign protein of increase genetic engineering.
Vertebrate cells, particularly mammalian cell, are also widely used for the expression of recombinant protein.In culture
The amount of the protein production of the cell of growth, over time, dependent on a number of factors, e.g., for example cell density, cell cycle phase,
The cell biological aggregate velocity of albumen, the longevity to sertoli cell vigor and the ambient condition and cell of growth in culture
Life (i.e. how long afterwards they die from apoptosis or apoptosis).For example by controlling nutrients, cell density, oxygen and two
Content, lactic dehydrogenase, pH, permeability, metabolin of carbonoxide etc., developed improve vigor of the cell in culture and
The various methods in life-span, and the method for improving the yield of required albumen.
Other host cells can be used for production heterologous recombinant protein, particularly plant cell and yeast cells.
Many pharmaceutical proteins of mammal source have been synthesized in plant.These such as have every from blood product
Year more than 500 tons of demands human serum albumins, to needing lesser amount of cell factor and other signaling molecules.It is most of to plant
The albumen in thing source is produced in transgene tobacco, and is directly extracted from leaf.Generally, these albumen are with relatively low
Level is produced, typically smaller than the 0.1% of total soluble protein.It is so low-level to produce the group for especially reflecting various factors
Close, most important of which is that protein folding and stability are poor.Recently, tobacco chloroplast system has been used for higher water-glass
Intelligent's albuminoid (MA JKC et al., 2004).
Yeast system is the main matter for producing a large amount of industry and bio-pharmaceuticals albumen for many years.Yeast is clear and definite
Very high cell quality density can be grown into the medium of definition.Recombinant protein can be over-expressed in yeast, so that product
Secrete, and can be reclaimed in zymotic fluid from cell.The albumen of yeast secretary, in consistent glycosylation site, by a large amount of glycosyls
Change.Therefore, expression of the recombinant protein in Yeast system, which is always restricted for post-translational glycosylation pattern, does not influence albumen work(
Those albumen of energy.Some yeast expression systems are used for expression of recombinant proteins, including Blastocystis (Sacharomyces), division
Yeast (Schizosaccharomyces pombe), Pichia pastoris (Pichia pastoris) and multiple-shaped nuohan inferior yeast
(Hansanuela polymorpha).Recently, a kind of the new of ability for having and recombinant glycoprotein being produced in yeast is occurred in that
System, its glycosylation sequences are similar to the secreting type human glucoprotein produced in mammalian cell.By eliminating endogenous enzyme (its
Intermediate addition high mannose chain is glycosylated to N-) modify the glycosylation approach of Pichia pastoris.In addition, participating in humanization oligosaccharides
At least five kinds organized enzymes of chain synthesis are specifically transfected to Pichia pastoris.A large amount of humanization glycoprotein are produced in yeast
Ability imparts such advantage, and glycosylation structure is high unity and is easy to purifying wherein.Furthermore, it is possible to by ferment
Using stream plus (fed-batch) production in mother, using the fermentation time more shorter than mammalian cell, to eliminate by mammal
The cross pollution of viral and other mammalian hosts glycoprotein.
However, by using these systems, heterologous protein, phase are produced with about 1-2mg/L in the supernatant of culture cell
Compared with industrial production purpose, this is at a fairly low.
Therefore, the system in the urgent need to providing a heterologous protein expression that can reach significantly higher level.
The present invention has catered to this needs, and there is provided protein expression, this method has been reached than existing protein production
The level of production (that is, in supernatant until 200mg/L albumen) that high 100 times of means.
The present inventor shows really, comes from people 6- methyl guanine-DNA- transmethylases (hMGMT) egg using coding
The nucleotide carrier of white albumen, the albumen in the hMGMT sources is direct or indirectly connects protein of interest, by the interest
The production of albumen is averagely enhanced to 40mg/L to 200mg/L yield.
Brief description of the drawings
Figure 1A discloses the schematic diagram for the mRNA for encoding MGMT fusion proteins sequence of the present invention, from 5' to 3', contains signal
Peptide, MGMT mRNA sequences, intervening sequence, proteolytic cleavage site, recombinant protein gene (foreign gene), intervening sequence and one
Individual mark (His6) label.
Figure 1B：The DNA and amino acid sequence of carrier same section, it includes i) insect ssBiP signal peptides (italic), ii)
SNAP coding enhancer sequence (grey), iii) DNA intervening sequences, iv) enterokinase site coded sequence (runic), v) clone position
Point EcoRV/XmaI (underscore) and the DNA (bold Italic) that vi) coding His labels are marked (are also shown in SEQ ID NO：5).
Fig. 2A and Fig. 2A (Continued) discloses fusion protein S NAP (grey) and is connected to the Rift Valley fever virus of His labels mark
The amino acid sequence of (Rift Valley fever virus) N nucleoprotein (RVF.N, runic), two kinds of albumen are by intervening sequence
GGGS is separated.
Fig. 2 B：There are SEQ ID NO in transfection：On the cell conditioned medium of the S2 cells of 19 (SNAP-RVF) DNA vector, with or
Stimulated 10 days without cadmium, use anti-HisLabelAntibody immunoblotting assay.
Fig. 2 C：With anti-His antibody, in insoluble (INS) or solvable (SOL) protein part of Escherichia coli B21 lysates
(fraction) immunoblotting assay carried out on, the bacterium carries pET302/RVF.N+proTEV+GST plasmids.
Fig. 2 D：Immunoblotting assay shows that the secretion chimeric protein SNAP-RVF.N of the S2 cells stimulated from 10 days exists
The amount of SNAP-RVF.N in continuous part (fraction) sample obtained after two-step purifying, using Talon and
The posts of Superdex 75.
What Fig. 3 A and Fig. 3 A (Continued) disclosed fusion protein S NAP (italic) and the envelope protein E from west nile virus can
Molten form (grey), is connected to the DNA and amino acid sequence of His labels mark (runic), and albumen is separated by intervening sequence GGGS
(SEQ ID NO：20).
Fig. 3 B：The immunoblotting assay carried out using anti-His tag antibodies, being shown in transfection has present invention coding SNAP-
WNsE DNA vector (SEQ ID NO：20) in the supernatant of S2 cells, stimulated 10 days with or without cadmium, Nile virus envelope
The secretion of the soluble form of albumen E protein.
Fig. 4 A disclose containing BiP peptide signals, MGMT samples coded sequence (SNAP samples), two in IFN α sequence both sides
The schematic diagram of the DNA boxes of pro-TEV cleavage sites (huIFNAI) and His labels mark.
Fig. 4 B and Fig. 4 B (Continued)：Fusion protein S NAP (grey, before be insect peptide signal that italic is represented) and IFN α are (thick
Body) DNA and amino acid sequence of (bold Italic), SNAP and IFN α albumen enterokinase cleavage site point are marked followed by His labels
(underscore) and intervening sequence GGGS are separated.
Fig. 4 C：The immunoblotting assay carried out using anti-His tag antibodies, to detect that transfection has present invention coding IFN α
(S2/SNAP-IFN) expression of IFN α in carrier or the S2 cell conditioned mediums of control vector, with or without Cd2+Stimulate.
Fig. 4 D：Using anti-SNAP antibody, the 10 μ L S2/DeSNAPuniv-IFN α cells of 10 days are being induced with or without cadmium
The immunoblotting assay carried out on supernatant.
Fig. 4 E：In the HeLa of the chikungunya virus (Chikungunya virus) infected with expression renilla luciferase
The activity of luciferase in cell, is produced with the from commercial source (Intergen) of various dose or by the method for the present invention
Raw IFN α handles the cell.
Fig. 4 F：The work of luciferase in the HeLa cells infected with the chikungunya virus of expression renilla luciferase
Property, handle the cell with the SNAP-IFN α albumen obtained by production method of the present invention of various dose.
Fig. 5 represents the different step in recombinant protein production process of the present invention.
Fig. 6 A and Fig. 6 A (Continued) discloses fusion protein S NAP (grey, before be insect peptide signal) and granzyme M is subsequent
The DNA and amino acid sequence of His labels, SNAP and granzyme M albumen with enterokinase cleavage site point and intervening sequence GGGS every
Open.
Fig. 6 B：Three potential GrM cleavage sites in chimeric fusion protein SNAP-GrM schematic diagram, prominent SNAP.
Fig. 6 C：The immunoblotting assay carried out using anti-SNAP or anti-His tag antibodies, to detect that transfection has volume of the present invention
Code GrM (S2/SNAP-GrM, SEQ ID NO：55) SNAP-GrM expression in the S2 cell conditioned mediums of carrier.
Fig. 7 A are disclosed to be cut containing BiP samples peptide signal, MGMT coded sequences, two pro-TEV in IFN α sequence both sides
Cut the schematic diagram of the general DNA boxes of site (huIFNAI) and His labels mark.
Fig. 7 B and Fig. 7 B (Continued)：Fusion protein S NAP (grey, before be insect Bip samples peptide signal) and (amino of people's IFN α 1
Acid is shown in bold) DNA and amino acid sequence that are marked followed by His labels, SNAP and IFN α albumen proTEV cleavages
Point and intervening sequence GGGS are separated.
Fig. 7 C：The immunoblotting assay carried out with anti-SNAP antibody, to detect that transfection has separately encoded SNAP without peptide
The carrier (pSNAPf carriers) of signal or separately encoded SNAP, are the carrier (pDV1ssprM- of dengue virus peptide signal before it
SNAP) or coding IFN α include DNA sequence dna as defined in Fig. 7 A carrier of the present invention (pDeSNAP-4/SNAP-IFNA1,
SEQ ID NO：57) SNAP-IFN α expression in HeLa cell conditioned mediums.
Fig. 8 A are disclosed comprising BiP samples peptide signal, SNAP coded sequences, two pro-TEV cleavage sites, His label marks
Remember, for cloning four of interest genes unique cloning site BamHI, Eco RV, Xma I and Apa I and intervening sequence
GGGS general DNA boxes (DeSNAP univ, SEQ ID NO：59 and 60).Need unique site Nhe I and 3' ends at 5' ends
The subcloning steps (such as plasmid pcDNA3 or pCI-neo) that Not I/Hind III are used in mammalian expression vector, and
Needing the Age I at unique site Bgl II and 3' ends at 5' ends is used for the subcloning steps of spinal animal DES systems.
Schematic diagram in Fig. 8 B disclose comprising BiP samples peptide signal, MGMT coded sequences, two pro-TEV cleavage sites,
His labels mark, for clone four of interest genes unique cloning site BamHI, Eco RV, Xma I and Apa I and
Intervening sequence GGGS general DNA boxes (DeMGMT univ, SEQ ID NO：69 and 70).
Fig. 9 discloses a kind of mode that foreign gene is inserted to DeMGMT Univ.
Figure 10 A disclose the SNAP fusion proteins CHIK.sE2- of the incubation 4 days at -80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C
SNAP, SNAP-WN.EDIII heat endurance.
Figure 10 B disclose the SNAP fusion protein S NAP-IFN α I of incubation 4 days at -80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C
Heat endurance.
Figure 10 C：Be incubated at -80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C bimestrial SNAP fusion proteins CHIK.sE2-SNAP,
SNAP-WN.EDIII heat endurance.
Figure 10 D：Bimestrial SNAP fusion protein Ss NAP-IFN α I heat is incubated at -80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C
Stability.
Figure 11 A are disclosed by the way that the carrier of the present invention is introduced into S2 cells, after being induced 10 days with (+) or without (-) cadmium,
Fusion protein S NAP-SSX2 and SNAP-sFasL production in whole supernatants.
Figure 11 B are disclosed by the way that the carrier of the present invention is introduced into S2 cells, after being induced 10 days with (+) or without (-) cadmium,
The fusion protein S NAP-sFasL production in different piece.
Figure 11 C are disclosed by the way that the carrier of the present invention is introduced into S2 cells, after being induced 10 days with (+) or without (-) cadmium,
The fusion protein S NAP-SSX2 production in different piece.
Figure 12 A disclose comprising BiP samples peptide signal, MGMT coded sequences (SNAP samples), two of SSX2 cancer antigens both sides
The schematic diagram of the general DNA boxes of pro-TEV cleavage sites and His labels mark.
Figure 12 B：Comprising BiP samples peptide signal, MGMT coded sequences, NERMCSL albumen both sides two pro-TEV cleavages
The schematic diagram of the general DNA boxes of point and His labels mark.
Figure 12 C：Using the anti-SNAP antibody of mouse, the Western blotting carried out on the HeLa cells of transient transfection two days divides
Analysis, the extracellular or intracellular production of display IFN α, SSX2 and NERMCSL.
Figure 13 A are disclosed comprising BiP samples peptide signal, MGMT coded sequences (SNAP samples), hSULF-2ΔTMDPolypeptide both sides
The schematic diagram of the general DNA boxes of two pro-TEV cleavage sites and His labels mark.
Figure 13 B and Figure 13 B (Continued)：Fusion protein S NAP (Dark grey, before be insect BiP samples peptide signal) and hSULF-2ΔTMDThe DNA and amino acid sequence marked followed by His labels, SNAP and hSULF-2ΔTMDAlbumen with proTEV cleavage sites and
Intervening sequence GGGS is separated.
Figure 13 C：Transient transfection has pcDNA3/DeSNAPuniv-hSULF-2ΔTMDSecreted by the cells of HEK 293 of two days
Secreting type is fitted together to DeSNAP-hSULF-2ΔTMDEnzymatic activity.
Figure 14 A disclose comprising BiP peptide signals, MGMT coded sequences (SNAP samples), two of NERMCSL albumen both sides
The schematic diagram of the DNA boxes of pro-TEV cleavage sites and His labels mark.
Figure 14 B：The immunoblotting assay carried out using anti-SNAP antibody, to detect that transfection has present invention coding NERMCSL
The carrier (S2/SNAP-NERMCSL) of albumen or coding chikungunya virus soluble protein E2 carrier (CHIK.sE2-
SNAP in S2 cell conditioned mediums), with or without Cd2+Stimulate, the expression of NERMCSL albumen.
Embodiment
The inventor have observed that 6- methyl guanines-dnmt rna (MGMT) and interest recombinant protein being total to together
Expression drastically increases the recombinant protein in insect cell such as S2 cells and in mammalian cell such as HeLa cells
Production.
6- methyl guanine-DNA- transmethylases (MGMT, also referred to as Atase or AGT, hereinafter referred to as " MGMT ") exist
Numbering is EC 2.1.1.63 in IUBMB enzyme nomenclatures.It is the 6- alkylguanine-DNA- alkyl turn of 207 amino acid residues
The DNA repair enzymes of enzyme are moved, its function in the cell is to repair alkylation DNA.More accurately, MGMT is by by SNIn 2 reactions
Methyl be converted into reactive cysteine residues (cysteine 145) to the O in DNA6- the guanine that methylates plays a role.
Because albumen is irreversibly inactivated, the repair mechanism is uncommon (Pegg A.E. et al., Mutat.Res.2000；462,
82-100).At present in molecular biology, by with O6The irreversible mark reaction of-benzyl guanine derivative, this enzyme is used
To mark upper report molecule (Juillerat A. et al., Chemistry＆Biology, vol.10,313- in vivo to albumen
317,2003 and WO 2005/085470).
Up to the present, it has been described that (Lim A. et al., EMBO are J.15 for the different enzymes from MGMT:4050-4060,
1996；Daniels D.S. et al., EMBO are J.19:1719-1730,2000；Juillerat A. et al., Chemistry＆
Biology,vol.10,313-317,2003、WO 2005/085470、WO 2004/031405).Especially, contained
Be mutated Cys62Ala, Lys125Ala, Ala127Thr, Arg128Ala, Gly131Lys, Gly132Thr, Met134Leu,
Arg135Ser, Cys150Ser, Asn157Gly, Ser159Glu, the albumen (WO for the 20kDa being truncated in 182 amino acids
" AGT26 " mutant so-called in 2005/085470, be also referred to as in WO 2006/114409 " SNAP 26 ").Specific mutation
Body " SNAP26 " is had been demonstrated with enhanced mark activity.However, never display or there is no suggestion that mistake, it can strengthen and it is even
The expression of the recombinant protein of connection.
Herein the present inventor propose first using MGMT enzymes (EC 2.1.1.63), its mutant, its catalyst structure domain or its
Sub-piece, for strengthening production of the albumen in host cell, particularly in spinal animal and vertebrate host cell.
When host cell expression includes at least i) having functional peptide secretion signal, ii in the host cell) MGMT enzymes, its mutation
Body, catalyst structure domain or sub-piece and iii) protein of interest fused polypeptide when, it was observed that humidification.In order that enhancing is made
With generation, MGMT enzymes directly or indirectly (can introduce intervening sequence and other amino acid) and be physically connected to protein of interest.Not by
Theory constraint, it is contemplated that MGMT enzymes can be as chaperone, such as by beneficial to the secretion from host cell and in host
The fused polypeptide of stable synthesis in cell conditioned medium, or prevent during synthesizing and secrete from host cell and be metabolized afterwards
Fall.
Further it has been observed that, MGMT has three-dimensional globular structure, and it includes α spirals (Wibley JEA et al., 2000),
This is mutually compatible with MGMT support effect.
In the context of the present invention, " host " cell is any cell that can be used for production recombinant protein, such as " spinal
Animal " (or invertebrate) cell, vertebrate cells, plant cell, yeast cells or prokaryotic.Preferably, they
It is non-vertebrate and vertebrate cells.
Spinal animal (also referred to as invertebrate) includes different doors, most it is well known that insect, Arachnoidea
(Arachnida), Crustachia (Crustacea), Mollusca (Mollusca), Annelida (Annelida), cirrus
Guiding principle (Cirripedia), Radiata (Radiata), Coelenterata (Coelenterata) and Ciliata
(Infusoria).They are divided into more than 30 door now, from simple organism, such as sponge and flatworm to complicated animal, such as
Arthropod and mollusk.In the context of the present invention, spinal zooblast is preferably insect cell, such as drosophila or mosquito
Daughter cell, more preferably Drosophila S 2 cells.
Can be used as host cell line the cell example from vertebrate organism include non-human embryonic stem cell or
Its derivative, such as fowl EBX cells；Conversion has the monkey kidney CVI systems (COS-7, ATCC CRL 1651) of SV40 sequences；Human embryo
Kidney system (293)；Baby hamster kidney cell (BHK, ATCC CCL 10)；Chinese hamster ovary cell (CHO)；Mouse sertoli cells
[TM4]；MK cells (CVI, ATCC CCL 70)；African green monkey kidney cell (VERO-76, ATCC CRL-1587)；People's uterine neck
Cancer cell (HeLa, ATCC CCL2)；MDCK (MDCK, ATCC CCL 34)；Buffalo rats liver (BRL 3A,
ATCC CRL 1442)；Human pneumonocyte (W138, ATCC CCL 75)；Human liver cell (Hep G2, HB 8065)；Mammary gland of mouse swells
Oncocyte (MMT 060562, ATCC CCL51)；Rat hepatoma cell [HTC, Ml.5]；YB2/O(ATCC n°CRL1662)；
NIH3T3；HEK and TRI cells.In the context of the present invention, vertebrate cells be preferably EBX, CHO, YB2/O, COS,
HEK, NIH3T3 cell or derivatives thereof.
In the context of the present invention, available plant cell be tobacco cultivars Bright Yellow 2 (BY2) and
Nicotiana tabacum 1 (Nicotiana Tabaccum) (NT-1).
In the context of the present invention, available yeast cells is：Saccharomyces cerevisiae (Saccharomyces
Cerevisiae), fusion yeast (Schizosaccharomyces pombe) and multiple-shaped nuohan inferior yeast (Hansanuela
), and (methylotropic yeast) methanotrophic yeast such as Pichia pastoris and pichia methanolica polymorpha
(Pichia methanolica)。
In the context of the present invention, available prokaryote is typically E. coli bacteria or bacillus subtilis
(Bacillus Subtilis) bacterium.
Therefore, at least a) preferably there is work(in spinal zooblast or vertebrate cells the invention discloses coding
The peptide secretion signal of energy and b) 6- methyl guanines-DNA- transmethylases, its mutant, sub-piece or catalyst structure domain
Polynucleotide expression vector.
Term " carrier " is referred to herein as the DNA or RNA sequence of foreign gene can be introduced into host cell to turn whereby
Change and promote the medium of the expression of introduced sequence.Carrier may include for example, plasmid, bacteriophage and virus, and below more
It is discussed in detail.In fact, any kind of plasmid, clay, YAC or viral vector can be used for preparing and being introduced into host cell
Recombinant nucleic acid construct, and in host cell obtain protein of interest expression.Or, wherein it is desirable in certain types of host
When expressing protein of interest in cell, the viral vector of cell type or organization type needed for selectivity infection can be used.At this
In the context of invention, it is also important to note that the carrier for gene therapy (that is, can be to HOST ORGANISMS delivering nucleic acid point
Son).
For example, viral vector such as slow virus, retrovirus, herpesviral, adenovirus, adeno-associated virus, poxvirus, bar
The viral and other recombinant virus with required cell tropism of shape.For build and using viral vector method in the art
It is known (referring to Miller and Rosman, BioTechniques, 7:980-990,1992).
Actual preferred viral vector is highly suitable in vertebrate and spinal zooblast in the present invention
Those.
For spinal zooblast, carrier preferably is arboviruse, particularly preferred west nile virus, and it is that segmental appendage is moved
Thing carrier.Known is baculoviral by other carriers of effective expression in spinal zooblast.
For vertebrate cells, preferably slow virus, AAV, baculoviral and adenovirus vector.Suitable in mammal place
The carrier expressed in chief cell can also be non-viral (such as DNA) source.Suitable plasmid vector includes but is not limited to
PREP4, pCEP4 (Invitrogene), pCI (Promega), pCDM8 and pMT2PC, pVAX and pgWiz.
For prokaryote, preferred plasmid, bacteriophage and cosmid vector.Suitable carrier for prokaryotic system
Including but not limited to pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), p Poly,
pTrc；pET 11d；pIN；With pGEX carriers.
For plant cell, preferred plasmid expression vector such as Ti-plasmids, and virus expression carrier such as cauliflower mosaic virus
And tobacco mosaic virus (TMV) TMV (CaMV).
Expression of the recombinant protein in yeast cells can be implemented by the carrier of three types：Integration vector (YIp),
Plasmid episomal (YEp) and centriole plasmid (YCp)：Suitable carrier for the expression in yeast (such as saccharomyces cerevisiae) includes
But it is not limited to pYepSec1, pMFa, pJRY88, pYES2 (Invitrogen Corporation, San Diego, Calif.)
With pTEF-MF (Dualsystems Biotech production code members:P03303).
The carrier that can be used for gene therapy is known in the art.They are such as slow virus, retrovirus, adenopathy
Poison, poxvirus, herpesviral, measles virus, foamy virus or adeno-associated virus (AAV).Viral vector, which can have, replicates energy
Power, or can not have replication capacity in heredity, so that as replication defective or that duplication is impaired.It is preferred that gene therapy carry
Body is the DNA Flap carriers described in WO 1999/055892, US 6,682,507 and WO2001/27300.
The sequence of " coding " expression product (such as RNA, polypeptide, albumen or enzyme) is, when expression, to cause RNA, polypeptide, egg
The nucleotide sequence that white or enzyme is produced；That is the amino acid sequence of nucleotide sequence " coding " RNA or its coded polypeptide, albumen or enzyme
Row.
In the context of the present invention, " catalyst structure domain " of enzyme refers to the avtive spot of enzyme, or, in other words, occur
The enzyme molecule part of substrate catalysis is (herein, in SNIn 2 reactions, methyl group is converted into reactive cysteine residues).Cause
This, term " its catalyst structure domain " is appointed as any fragment or homologous sequence of MGMT polypeptides, preferably with least 80% day
The catalytic activity of right MGMT enzymes.These fragments (also referred to as " sub-piece ") may include between 20 to 180, preferably 30 to 100
Between amino acid.The homologous sequence of the catalyst structure domain can cause the part of the catalytic activity with one or more
Or the mutation all lost.
In the context of the present invention, MGMT enzymes can be sequence SEQ ID NO：4 people MGMT (refers to NP_
002403.2) NP_032624.1 mouse MGMT (SEQ ID NO, are accredited as：45), it is accredited as NP_036993.1 rat
MGMT(SEQ ID NO：Or its homologous sequence 46).
Term " homologous " refers to the sequence with sequence similarity.Term " sequence similarity ", with its all grammer shape
Formula, refers to the homogeneity or degree of correspondence between nucleic acid or amino acid sequence.In the context of the present invention, when at least about
80% or at least about 81% or at least about 82% or at least about 83% or at least about 84% or at least about
85% or at least about 86% or at least about 87% or at least about 88% or at least about 89% or at least about
90% or at least about 91% or at least about 92% or at least about 93% or at least about 94% or at least about
95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% amino acid is similar
When, two amino acid sequences are " homologous ".It is preferred that, it is similar or homologous by using the identification of Needleman with Wunsch algorithms
Peptide sequence.
Preferably, and 6- methyl guanines-homologous sequence of dnmt rna and SEQ ID NO：4 share at least
64% amino acid sequence identity, preferably at least about 65% amino acid sequence identity or at least about 66% amino
Acid sequence identity or at least about 67% amino acid sequence identity or at least about 68% amino acid sequence are same
Property or at least about 69% amino acid sequence identity or at least about 70% amino acid sequence identity or at least
About 71% amino acid sequence identity or at least about 72% amino acid sequence identity or at least about 73% ammonia
Base acid sequence identity or at least about 74% amino acid sequence identity or at least about 75% amino acid sequence are same
One property or at least about 76% amino acid sequence identity or at least about 77% amino acid sequence identity or extremely
Few about 78% amino acid sequence identity or at least about 79% amino acid sequence identity or at least 80%
Amino acid sequence identity or at least about 81% amino acid sequence identity or at least about 82% amino acid sequence
Homogeneity or at least about 83% amino acid sequence identity or at least about 84% amino acid sequence identity or
At least about 85% amino acid sequence identity or at least about 86% amino acid sequence identity or at least about 87%
Amino acid sequence identity or at least about 88% amino acid sequence identity or at least about 89% amino acid sequence
Row homogeneity or at least about 90% amino acid sequence identity or at least about 91% amino acid sequence identity or
The amino acid sequence identity or at least about 93% amino acid sequence identity of person at least about 92% or at least about
94% amino acid sequence identity or at least about 95% amino acid sequence identity or at least about 96% amino
Acid sequence identity or at least about 97% amino acid sequence identity or at least about 98% amino acid sequence are same
Property and or at least about 99% amino acid sequence identity.In one preferred embodiment, SEQ ID NO：4 it is same
Source sequence and SEQ ID NO：4 at least 64%, preferably 70% and more preferably 80% are identical.
Preferred homologous MGMT sequences include the mutation described in WO 2005/085470, and its position can be easily
Exchange with based on SEQ ID NO：4, SNAP26 start methionine residue correspond to SEQ ID NO：The methionine of 4 the 32nd is residual
(therefore, 31 amino acid should be added in the position disclosed in WO 2005/085470 base, so as to obtain SEQ ID NO：In 4
It is corresponding those).
Preferably, the MGMT homologous sequences available for the present invention correspond to SEQ ID NO：4 wild type MGMT sequences, its
In between 1 and 30, between preferably 6 and 25, and especially 14,15,16,17,18,19,20,21,22 or 23 amino
Acid is by other 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, and/or 1 to 40, C- ends, preferably 1 to 20, especially 10 to 20 amino acid, more preferably
15 amino acid are deleted.
In one preferred embodiment, with SEQ ID NO：4 compare, and MGMT homologous sequences include following mutation：
(A) Lys31 is substituted by Arg or Met32 is substituted by that Ser or Cys93 is substituted by Ala or Lys156 is substituted by
Ala or Ala158 be substituted by Thr or Arg159 be substituted by Ala, Gly162 be substituted by Lys or Gly163 be substituted by Thr or
Met165 is substituted by Leu or Arg166 be substituted by Ser or Cys181 be substituted by Ser or Asn188 be substituted by Gly or
Ser190 is substituted by Glu or Gly214 be substituted by Pro or Ser215 be substituted by Ala or Ser216 be substituted by Gly or
Gly217 is substituted by Ile or Leu218 be substituted by Gly or Gly220 be substituted by Pro or Ala221 be substituted by Gly or
Trp222 is substituted by Ser, or
(B) Lys31-Met32 is substituted by Arg-Ser or Ala158-Arg159 is substituted by Thr-Ala or Gly162-
Gly163 is substituted by Lys-Thr or Met165-Arg166 is substituted by Leu-Ser or Gly162-Gly163/Met165-
Arg166 is substituted by Lys-Thr/Leu-Ser or Asn188/Ser190 is substituted by Gly/Glu or Gly214-Ser215-
Ser216-Gly217-Leu218 is substituted by Pro-Ala-Gly-Ile-Gly or Gly220-Ala221-Trp222 is substituted by
Pro-Gly-Ser, it is preferably combined with any other 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor described in (A), or
(C) blocked after Leu223 (amino acid 224-238 be deleted), preferably with it is any other described in (A) or (B)
49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is combined.
It is preferred that MGMT homologous sequences be those being truncated after Leu223.
It is preferred that MGMT homologous sequences be wherein occur modification (B) in 2 modification those, optionally after Leu223
Block.
It is preferred that MGMT homologous sequences be wherein occur modification (B) in 3 modification those, optionally after Leu223
Block.
It is preferred that MGMT homologous sequences be wherein occur modification (B) in 4 modification those, optionally after Leu223
Block.
It is preferred that MGMT homologous sequences be wherein occur modification (B) in 5 modification those, optionally after Leu223
Block.
It is preferred that MGMT homologous sequences be wherein occur modification (B) in 6 modification those, optionally after Leu223
Block.
Other preferred MGMT homologous sequences be containing 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,
18th, those of the combination of 19 or 20 mutation selected from the modification disclosed in (A), and optionally being blocked after Leu223.
In particular it is preferred that containing mutation Lys31Arg, Met32Ser, Cys93Ala, Lys156Ala, Ala158Thr,
Arg159Ala、Gly162Lys、Gly163Thr、Met165Leu、Arg166Ser、Cys181Ser、Asn188Gly、
Ser190Glu、Gly214Pro、Ser215Ala、Ser216Gly、Gly217Ile、Leu218Gly、Gly220Pro、
Ala221Gly, Trp222Ser and homologous sequence (that is, the SEQ ID NO blocked after Leu223：2 SNAP sequences).
In a preferred embodiment, MGMT enzymes are SEQ ID NO：2 SNAP mutains or its homologue.
SEQ ID NO：2 SNAP mutant and people's 6- methyl guanine-DNA- transmethylases (NP_002403.2, SEQ ID NO：
4) amino acid sequence shares 77% homology, with mouse 6- methyl guanine-DNA- transmethylases (NP_032624.1,
SEQ ID NO：45) amino acid sequence shares 70% homology.
Preferably, the homologous sequence of the SNAP albumen and sequence SEQ ID NO：2 SNAP albumen at least more than 80%,
It is preferred that 81%, more preferably 82%, more preferably 83%, more preferably 84%, more preferably 85%, preferably 86%, more preferably 87%, it is more excellent
Select 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably 94%, it is more excellent
Select 95%, more preferably 96% to even more preferably 97% identical.
Preferably, polynucleotide expression vector of the invention also encodes the allogeneic dna sequence of protein of interest including enabling
Carry out the cloning site of inframe insertion.
As represented in the present invention, term " peptide secretion signal " represents short (the 3-60 amino acid for instructing albumen to transport
It is long) peptide chain.
Include but is not limited to mating factor (MF) α signal peptide sequence (US suitable for the example of the secretion signal of the present invention
5,879,926)；Invertase (WO 84/01153)；PHO5(DK 3614/83)；YAP3 (yeast aspartic proteases 3；WO
95/02059)；With BAR1 (WO 87/02670).
In the context of the present invention, this peptide secretion signal preferably in spinal zooblast or vertebrate cells or
Person is tool functional among two kinds.
The example of tool functional peptide secretion signal is in insect cell：Insect ssBiP (SEQ ID NO：48, for example
With SEQ ID NO：11 DNA sequence dna), SEQ ID NO：51 BiP samples peptide signal is (such as with SEQ ID NO：50
DNA sequence dna) and arboviruse present in any peptide signal, envelope E protein (the SEQ ID NO of such as west nile virus：
15)。
It is interesting that above-mentioned BiP samples peptide signal is functional in spinal animal and vertebrate cells.It is this
BiP sample signals correspond to SEQ ID NO：48 BiP peptide signals, wherein last glycine amino acid correspond to dengue virus
Amino acid sequence Pro Thr Ala Leu Ala (the SEQ ID NO of E protein cleavage site：61) replace.Therefore, once albumen
It is translated and is secreted into host cell supernatant, BiP samples signal will be advantageously cut.
Various secretion signals can also be used for the expression in yeast host cell, such as in saccharomyces cerevisiae.This includes precursor α
The factor (prepro alpha factor), HSp150, PHO1, SUC2, KILM1 (the malicious toxin of 1 type killing) and GGP1.
Cloning site is will conveniently to encode sequence of the gene cloning of protein of interest into expression system.It contains restrictive
Site or restricted recognition site, i.e. the position on the DNA molecular containing specific nucleotide sequences, it is by Restriction Enzyme institute
Identification.These are typically palindromic sequence (because Restriction Enzyme usually as homodimer combination), and specific Restriction Enzyme
Sequence within its cleavable recognition site or between two nucleotides nearby.Cloning site is known to those skilled in the art
's.
It is further preferred that polynucleotide expression vector also includes the coding interest heterologous protein being inserted into the cloning site
Or the allogeneic dna sequence of heterologous polypeptide.
Term " heterologous " refers to the combination of non-naturally occurring element.For example, the present invention includes coding " protein of interest/many
" allogeneic dna sequence " of peptide ", these DNA sequence dnas are not natively in or positioned at the dye of the host cell for protein expression
In colour solid site.
It is excellent when in the nucleotide carrier that the allogeneic dna sequence for encoding interest heterologous protein or polypeptide is inserted into the present invention
Choosing require its coding include the signal peptide, the MGMT enzymes, its mutant or homologue and the interest heterologous protein/
The fused polypeptide of polypeptide.
In a preferred embodiment of the present invention, the DNA sequence dna for encoding the MGMT enzymes is located at the coding interest
The 5' or 3' of the DNA sequence dna of heterologous protein, preferably in 5'.Therefore, MGMT enzymes are connected directly or indirectly to interest heterologous protein/many
Peptide, is preferably placed at the N-terminal of interest heterologous protein/polypeptide.
Particularly preferably, when the active structure domain of interest heterologous protein/polypeptide is located at its timesharing of C- ends, such as IFN α, compile
The DNA sequence dna of its MGMT enzyme of code is located at the 5' for the DNA sequence dna for encoding the interest heterologous protein/polypeptide.According to identical
Mode, it can be particularly preferred, and when the active structure domain of interest heterologous protein/polypeptide is located at its N- ends timesharing, coding is described
The DNA sequence dna of MGMT enzymes is located at the 3' for the DNA sequence dna for encoding the interest heterologous protein/polypeptide.
More accurately, in the first aspect, it is used for the present invention relates to one kind in host cell, preferably in spinal animal
And/or in vertebrate host cell, more preferably in insect cell express recombinant protein carrier, the carrier include according to
5' to 3' direction encodes following nucleotide sequence in single ORFs：
A) have functional peptide secretion signal in the host cell,
B) 6- methyl guanines-DNA- transmethylases (MGMT, EC 2.1.1.63), its mutant or catalyst structure domain,
And
C) recombinant protein.
In the context of the present invention, term " recombinant protein " or " protein of interest " represent heterologous and produce celliferous base in albumen
Because of product or polypeptide, it is preferably selected from diagnosis and treatment albumen or polypeptide.
It is further preferred that the diagnosis and treatment albumen or polypeptide are selected from as follows：
- bacterium or the immunogenic protein of virus, the albumen of more preferably (infectious, pathogenic) virus, such as from stepping on
Leather, encephalitis B, tick propagate encephalitis, yellow heat, Usu figure, sieve Theo, Murray encephalitis, Wei Saiersi Blang (Wesselbron),
Hereby card or the EDIII albumen of west nile virus, or the nucleoprotein N from Rift Valley fever or toscana virus, or agree from datum hole
The soluble form of refined viral E2 envelope proteins, or west nile virus E envelope proteins soluble form, and
- blood factor, anticoagulant, growth factor, hormone, vaccine, therapeutic enzyme, monoclonal antibody and cell factor
(such as IFN-α, granzyme M and FasL),
The N- petiolareas of-antigen, such as cancer antigen, such as cancer testes antigens SSX2 or ERC/Mesotheline (no correspondence translation)
Domain (NERCMSL),
Sulfatase (heparan-sulfate6-O- in-anti-tumor protein, such as FasL, or heparin sulfuric acid 6-O-
Endosulfatases) (hSULF),
- microorganism, virus and/or parasite polypeptide,
- any other useful proteins (such as contactin).
Albumen FasL is a kind of pro apoptotic protein, and it may be used as antitumor agent.It can be for example by SEQ ID NO：88
Coding.
HSulf albumen (or hSULF) is internal regulation heparin sulfuric acid structure and growth and progress to malignant cell have
Sulfatase (Dai et al., 2005) in the heparin significantly affected-sulfuric acid 6-O-.In the context of the present invention, preferably hSulf2
Albumen, and more preferably hSulf-2ΔTMD, wherein membrane spaning domain (TMD) is deleted to improve its solubility, this mutant
With amino acid sequence SEQ ID NO：95, and by such as SEQ ID NO：94 codings.
The carrier of the present invention can also be used for expressing and purify interest peptide and/or polypeptide.In the context of the present invention, term
" peptide " and " polypeptide " is synonymous, represents the short polymer (also referred to as " residue ") of amino acid monomer being connected with peptide bond.These
Polymer preferably comprises the residue within the residue within 100, more preferably 50.
Particularly, carrier of the invention can be used for expressing and purifying diagnostic antimicrobial polypeptide, such as bacterium, virus or parasitic
Worm polypeptide.The example of this polypeptide is as secreted by bacterium, virus or parasite or Antigenic Peptide, mucoprotein and/or the poison of expression
Element.It is preferred that, the Antigenic Peptide is by influenza virus, hepatitis A virus, hepatitis type B virus, HCV, hepatitis G
Virus, inhibition of HIV, yellow fever virus, dengue virus, japanese encephalitis virus, Far East Russian encephalitis virus, Usu figure or Xi Niluo diseases
Poison, Rift Valley fever or toscana virus, chikungunya virus, Respiratory Syncytial Virus(RSV), rocio virus, Murray encephalitis viruses,
Wei Saiersi Blang virus, hereby card viral (Zika Virus), lymphocytic choriomeningitis (lymphocytic
Choriomeningitis) virus, human parvovirus, human papilloma virus, human cytomegalovirus or any disease identified
Poison expression.Preferably, the Antigenic Peptide by parasitic protozoa, (go into business by such as Entamoeba histolytica (Entamoeba histolytica), Lan Shi
Flagellate (Giardia lamblia)), worm (such as nematode, tapeworm or fluke) or arthropod (such as Crustachia, insect
Guiding principle, spider guiding principle) expression.It is preferred that, the Antigenic Peptide is by infective bacterial, such as streptococcus (Streptococcus), staphylococcus
Belong to (Staphylococcus) and Bacillus coli expression.Infectious prions are well known in the art.People can enumerate,
It is used as example, botulic neurotoxin, C. perfringens epsilon toxin, ricin (WA), saxitoxin, shiga toxin, fugutoxin
Element, staphylococcal enterotoxin etc..Mucoprotein is also known in the art.MUC5AC, MUC5B and MUC2 are the examples.These realities
It is not restricted to apply example, and any peptide/polypeptide can be expressed by the method for the present invention.
Contactin is the molecule subgroup of contactin, and it is expressed (see Shimoda in nervous system
And Watanabe, 2009 summary).They participate in phrenoblabia, especially self-closing disease.Produced by the system of the present invention
It is preferred that contactin is contactin 2 and contactin 4.For example contactin 4 (CNTN4) is by SEQ ID NO：91 codings are (right
Answer intact proteins NP_783200.1 amino acid/11 9-990).
Known a large amount of cancer antigens are the effective vaccine targets for treating cancer.A large amount of productions of this polypeptide are (see Cheever
Et al., the list in 2009) it is seemingly very important for obtaining effective cancer vaccine.It is interesting that the load of the present invention
Body results in the high-caliber restructuring cancer antigen available in immunization therapy or generation antibody or Method for cancer diagnostics.
SSX2 and NERCMSL are two examples of cancer antigen.SSX2 cancer antigens are by with SEQ ID NO：76 DNA encoding
(gene pool：NM_175698).ERC/Mesotheline N- petiolareas (NERCMSL) are by SEQ ID NO：83 codings.It is this anti-
Original is commonly used for suffering from the detection antigen in the patient of pernicious mesothelium.
The method according to the invention can produce any albumen.
It is preferable, however, that albumen is human cytokines, such as insulin, IFN, FasL, Mesotheline, hSULF or contact egg
In vain.
More generally, optimization protein is those for being up to the present difficult to largely produce.This albumen be such as FasL,
Granzyme M, hSULF, Mesotheline and contactin.
Coding includes the peptide signal, the MGMT enzymes, its mutant or catalyst structure domain and the interest recombinant protein
The DNA sequence dna of fused polypeptide can be operably coupled to inducible promoter, wherein inducible promoter is in identical host
Same with peptide signal in cell is functional.
It is highly preferred that in the carrier of the present invention, the ORFs is operably coupled to inducible promoter, its
It is functional that middle inducible promoter is same with peptide signal in identical host cell.
In cell, coded sequence " is operably coupled to " expression regulation sequence (i.e. transcription and translation regulating and controlling sequence),
When coded sequence is transcribed into RNA by RNA polymerase, RNA then carry out trans RNA montages (if it include introne) with
And, if the sequential coding albumen, translates into the albumen.
" promoter " is that the nucleotide sequence that a kind of thus starting transcription is operably coupled to DNA downstream (exists
The positive-sense strand 3' directions of double-stranded DNA).Visible transcription initiation site is (for example, can by using s1 nuclease mapping in promoter sequence
To be able to conveniently find), and the protein binding domain (consensus sequence) that responsible RNA polymerase is combined.
It can be used for the promoter of control gene expression in the context of the present invention for example in spinal zooblast or vertebra
It is functional in zooblast.For example, for spinal zooblast, the regulatory sequence of metallothionein gene can be used
(Brinster et al., Nature, 296:39-42,1982).
The inducible promoter in carrier of the present invention be preferably, there are in insect cell, more preferably in drosophila cell
In, with promoter activity.For example drosophila metallothionein promoter (Lastowski-Perry et al.,
J.Biol.Chem.260:1527 (1985))), it instructs high-caliber genetic transcription in the presence of metal such as copper sulphate.Or,
Drosophila Actin 5C gene promoters can be used, this is a kind of constitutive promoter and need not add metal
(B.J.Bond et al., Mol.Cell.Biol.6:2080(1986)).The example of other known drosophila promoter includes for example luring
Conductivity type heat shock (Hsp70) and COPIA LTR promoters.SV40 early promoters provide the table lower than drosophila metallothionein
Up to level.
The inducible promoter in carrier of the present invention be preferably, there are in Drosophila melanogaster cell, preferably in drosophila S2
There is promoter activity in cell.For example in Lastowski-Perry et al., J.Biol.Chem.260:Filled in 1527 (1985)
Divide the metallothionein promoter of description.
Promoter suitable for the constitutive expression in mammalian cell includes cytomegalovirus (CMV), and early stage starts immediately
Son, adenovirus major late promoter, phosphoglyceric kinase (PGK) promoter, the thymidine of herpes simplex virus (HSV) -1 swash
Enzyme (TK) promoter.The induction type eukaryotic promoter adjusted by the compound of exogenous offer includes but is not limited to the gold of zinc induction
Category sulfoprotein (MT) promoter, dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerases are opened
Subsystem (WO 98/10088), insect moulting hormones promoter, tetracycline repressible type promoter, tetracycline-inducible start
Son, RU486 inducible promoters and rapamycin inducible promoter.
The promoter in carrier of the present invention be preferably, there are in mammalian cell, preferably had in HeLa cells
Promoter activity.Such as promoters of SV 40.
A series of Yeast promoters can be used for the expressing protein in yeast host cell.Some such as ADH2, SUC2 are to lure
Conductivity type, and others such as GAPDH is composing type in expression.Other promoters suitable for being expressed in yeast include
TEF, PGK, MF α, CYC-1, GAL-1, GAL4, GAL10, PHO5, glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH) and ethanol
Dehydrogenase (ADH) promoter.
In order to in plant cell, the most frequently used promoter be cauliflower mosaic virus (CaMV) 35S promoter or its
Enhanced edition, but some can be used to substitute promoter, hybridization (ocs) 3mas such as from corn and arabidopsis (A.Thaliana)
Promoter or ubiquitin promoter.Different from these constitutive promoters, rice alpha-amylase RAmy3D promoters are deprived by sugar
Induce (Hellwig S et al., 2004).
Promoter suitable for being expressed in e. coli host cell includes but are not limited to bacteriophage lamba pL startups
Son, lac, TRP and IPTG induction type pTAC promoters.
It is preferred that, peptide secretion signal and inducible promoter are functionals in identical host cell.
It is further preferred that peptide secretion signal and inducible promoter are functional in Drosophila S 2 cells and vertebrate cells
's.
Term " induction type ", is well known to the skilled person when for promoter.Substantially, in induction
Expression under the control of type promoter, when responding applied stimulation, is turned " on " or increases.The property of stimulation is with promoter
It is different.It is suitably stimulate in the case of, expression that some inducible promoters cause seldom or to can't detect is (or not
Expression).In the case of no stimulation, other inducible promoters cause detectable constitutive expression.Do not stimulating
In the case of, regardless of expression, when there is correct stimulate, the expression from any inducible promoter is increase
's.
Once build appropriate carrier and be transfected into selected host cell, preferably drosophila cell line, by adding
Derivant suitable for inducible promoter carrys out the expression of inducing heterogenous albumen.Such as cadmium or copper are the derivants of Hsp70 promoters.
For constitutive promoter, such as actin 5C promoters, expression does not need derivant.
The people MGMT enzymes of the present invention are preferably by people's mgmt gene sequence NM_002412.3, (the SEQ ID of gene I/D 4255
NO：3) encode or by optimization SEQ ID NO：68 (including only 50%G/C) are encoded.However, in the context of the present invention
Its any homologous sequence can be used, as long as its encoding function MGMT enzymes, its mutant or catalyst structure domain, preferably SEQ ID
NO：4 or SEQ ID NO：2.
The preferred DNA sequence dna for encoding the MGMT mutant is SNAP DNA sequence dna SEQ ID NO：1, or coding
SEQ ID NO：The 2 but DNA sequence dna SEQ ID NO with 51%G/C contents：47 or SEQ ID NO：67.
In yet another embodiment of the present invention, a fragment of nucleotide carrier of the invention coding at least MGMT enzymes
(such as SEQ ID NO：4 fragment), or its homologue fragment (such as sequence SEQ ID NO：The piece of 2 MGMT mutant
Section), the factor of its at least 0.5 times of level increase for remaining to obtain than the total length enzyme originated with the fragment increases interest egg
The bioactivity of white expression.As an example, if utilizing SEQ ID NO：The level of production of 4 total length enzyme is 100mg/
L, then the SEQ ID NO with least 50mg/L levels of production：4 any fragment is (with SEQ ID NO：4 total length enzyme identicals
Experiment condition) it is included within the present invention.
In yet another embodiment of the present invention, polynucleotide expression vector encodes at least one peptide cleavage site, and its is excellent
Bit selecting is between MGMT enzymes or its catalyst structure domain and interest recombinant protein.
The cleavage site (also referred to as " peptide cleavage site ") of peptide be by least one protease (for example, serine protease,
Cysteine proteinase etc.) amino acid sequence that is recognized.The example of peptide cleavage site is SEQ ID NO：62 enterokinase is cut
Site (AspAspAspAspLys/Asp) is cut, such as by DNA sequence dna SEQ ID NO：Coded by 12.Enterokinase is a kind of silk
Serine protease (EC 3.4.21.9), it is known that nonactive trypsinogen is converted to activity by it by the C- ends of cutting sequence
Trypsase：Val--(Asp)4-- Lys--Ile--Val~(trypsinogen) → Val-- (Asp)4-- Lys (hexapeptide)+
Ile--Val~(trypsase).If before Lys being four Asp and not having proline residue below, enterokinase is relying
Cut after propylhomoserin.
Another useful peptide cleavage site is so-called " TEV protease " cleavage site, and it has amino acid sequence SEQ
ID NO：53 or SEQ ID NO：65 (Glu Asn Leu Tyr Phe Gln Gly or Ser), it is for example by DNA sequence dna SEQ
ID NO：52 or SEQ ID NO：66 codings.TEV protease is marmor erodens (tobacco etch virus) coding
The common name of the 27kDa catalyst structure domains of nuclear inclusion body albumen.It is commercially available (Invitrogen).
Cleavage site (the SEQ ID NO of film precursor prM from dengue virus serotypes 1：61) it can also be used for the present invention
Carrier in.
In another embodiment, polynucleotide expression vector of the present invention also encodes a mark, and it is preferably placed at this hair
The C-terminal (including peptide signal, mgmt protein or its homologue and recombinant protein) of recombinant protein in bright fused polypeptide.
In the context of the present invention, " mark " dedicated for being easy to reclaim polypeptide from the thick lysate of host cell,
And be preferably selected from：Fluorescin, polyhistidine (poly-his) or polyhistidyl-glycine (poly-his-gly) label；
Flu HA labels；C-myc label herpes simplex virus glycoproteins D (gD) label, Flag- peptides, alpha-tubulin epitope or T7 bases
Because of 10 protein peptide tags.However, it is possible to use any other mark.In a preferred embodiment of the present invention, carrier bag
Including coding has SEQ ID NO：14 six histidine-tagged DNA.
In another embodiment, polynucleotide expression vector of the invention also encoded interval sequence, is preferably placed at MGMT
Enzyme (or its catalyst structure domain) is between interest recombinant protein and/or between interest recombinant protein and mark.
In the context of the present invention, intervening sequence is to include the amino acid sequence of at least three amino acid, dedicated for
Space separates the polypeptide (and then these polypeptides are connected indirectly) of two connections.Such intervening sequence can be such as ammonia
Base acid sequence Gly-Gly-Gly-serine (GGGS, SEQ ID NO：63), and its DNA intervals sequence is encoded
Row are SEQ ID NO：13.In the context of the present invention, hereafter this DNA sequence dna is referred to as " DNA intervening sequences " and positioned at volume
Between code MGMT or the DNA and recombinant DNA sequence of its catalyst structure domain, the upstream of the DNA sequence dna of optimized encoding peptide cleavage site.
Polynucleotide expression vector disclosed in this invention can have sequence SEQ ID NO：9th, sequence SEQ ID NO：10 or
SEQ ID NO：64 (empty carriers that correspond to be not inserted into interest recombination in cloning site).In embodiment
In, carrier of the invention can be encoded：
- as described above peptide BiP insect signals (being functional preferably in S2 drosophila cells) or BiP sample signals,
-SEQ ID NO：4 mgmt protein or SEQ ID NO：2 SNAP albumen,
- interest recombinant protein,
- as described above enterokinase peptide cleavage site or proTEV cleavage sites,
- polyhistidine is marked, and
- there is amino acid sequence Gly-Gly-Gly-serine (GGGS, SEQ ID NO：63) between two
Every sequence.
In a preferred embodiment, expression vector codes of the invention：
-SEQ ID NO：48 peptide BiP insect signals,
-SEQ ID NO：4 mgmt protein or SEQ ID NO：2 SNAP albumen,
- interest recombinant protein,
-SEQ ID NO：62 enterokinase peptide cleavage site,
- polyhistidine is marked, and
- two intervening sequences with amino acid sequence Gly-Gly-Gly-serine (GGGS).
Another preferred embodiment in, expression vector codes of the invention：
-SEQ ID NO：51 BiP sample peptide signals,
-SEQ ID NO：4 mgmt protein or SEQ ID NO：2 SNAP albumen,
- interest recombinant protein,
-SEQ ID NO：53 proTEV peptide cleavage sites,
- polyhistidine is marked, and
- two intervening sequences with amino acid sequence Gly-Gly-Gly-serine (GGGS).
For example, such carrier can include sequence SEQ ID NO：19 (when protein of interest is RVF virus nucleoproteins N
When), SEQ ID NO：20 (when protein of interest is west nile virus nucleoprotein N), SEQ ID NO：21 or 57 or 72 or 74 (when
When protein of interest is IFN α), SEQ ID NO：77th, 79 or 81 (when protein of interest is cancer antigen SSX2), SEQ ID NO：55
(when protein of interest is granzyme M), SEQ ID NO：89 (when protein of interest is FasL), SEQ ID NO：84 or 86 (when
When protein of interest is cancer antigen NERCMSL) or SEQ ID NO：92 (when protein of interest is contactin CNTN4).
In second aspect, the invention also discloses the carrier for expressing recombinant protein in host cell, it includes
The following nucleotide sequence of direction encoding in single ORFs from 5' to 3'：
A) peptide secretion signal,
b)SEQ ID NO：4 mgmt protein or SEQ ID NO：2 SNAP albumen,
C) at least one peptide cleavage site,
D) polyhistidine is marked, and
E) at least one intervening sequence.
In one preferred embodiment, the peptide secretion signal is SEQ ID NO：50 BiP sample peptide signals.
In a preferred embodiment, the carrier includes two SEQ ID NO：52 proTEV peptide cleavages
Put and/or two have amino acid sequence SEQ ID NO：63 intervening sequence.
In a specific preferred embodiment, the carrier includes sequence SEQ ID NO：59 or SEQ ID NO：
69, the sequence is hereinafter referred to as general DeSNAP boxes " DeSNAP Univ " and DeMGMT boxes " DeMGMT in this application
Univ”。
These " DeSNAP Univ " (SEQ ID NO：59) with " DeMGMT Univ " (SEQ ID NO：69) it is referred to as " logical
With " sequence, because to produce heterologous protein in any kind of carrier dedicated for transfecting host, i.e., they be inserted into
Vertebrate carrier (such as pcDNA3 or PCI-neo carriers) and the spinal animal carrier (pMT/BiP/ as being used for DES systems
V5-HisA, is shown in following examples).
Plasmid example including the universal sequence is SEQ ID NO：64 (pUC57 for including DeSNAP Univ) and SEQ
ID NO：71 (pUC57 for carrying DeMGMT Univ).
Once the heterologous sequence of protein of interest is cloned here, such carrier can advantageously be transfected into vertebrate or
In spinal animal host cell, to produce a large amount of protein of interest.
In a third aspect, the present invention has the weight of the DeSNAP Univ or DeMGMT Univ carriers for stable transfection
Group cell, i.e. transfection have the expression vector for being included in and following nucleotide sequence being encoded in single ORFs, from 5' to 3'
Direction：
A) peptide secretion signal,
b)SEQ ID NO：4 mgmt protein or SEQ ID NO：2 SNAP albumen,
C) at least one peptide cleavage site,
D) polyhistidine is marked, and
E) at least one intervening sequence,
Each composition is as defined above.
It preferably includes SEQ ID NO：64 plasmid (pUC57 for including DeSNAP Univ) or SEQ ID NO：71 (bands
Have DeMGMT Univ pUC57) or at least nucleotide sequence SEQ ID NO：59 (DeSNAP Univ) or SEQ ID NO：69
(DeMGMT Univ)。
Preferably, in this aspect of the present invention, the recombinant cell is Bacillus coli cells.
Using this recombinant cell to expand and purify the expression vector of the present invention, SEQ ID NO are preferably included：59
DeSNAP Univ (such as SEQ ID NO：Or SEQ ID NO 64)：69 DeMGMT Univ (such as SEQ ID NO：71) those.
Therefore, present invention is alternatively directed to this recombinant cell, for producing any expression vector of the invention, (carrier is such as
It is upper described) purposes.
The polynucleotide expression vector of the present invention may also comprise coding selection marker thing and/or the gene of terminator sequence.
The selectable marker gene that construct can be included generally is to confer to those of selective meter's type, such as antibiotic resistance
(such as blasticidin S, ampicillin, kanamycins, hygromycin, puromycin, chloramphenicol).
In fourth aspect, the present invention relates to fused polypeptide, it is included in host cell, preferably in spinal animal or
In vertebrate cells, the functional peptide secretion signal more preferably in insect cell, and 6- methyl birds as described above are fast
Purine-DNA- transmethylases (MGMT) (EC 2.1.1.63), its mutant or catalyst structure domain.
In this fused polypeptide, preferably described MGMT enzymes are SEQ ID NO：4 albumen, SEQ ID NO：2 SNAP
Protein mutant or its homologue.
This fused polypeptide preferably also includes interest recombinant protein as described above, is preferably placed at MGMT enzymes as described above
Or the C-terminal of its catalyst structure domain, and/or mark.This mark is preferably polyhistidine mark, and is preferably placed at interest restructuring
The C-terminal of albumen.
The fused polypeptide of the present invention can be SEQ ID NO：33 to 43, SEQ ID NO：56 or SEQ ID NO：58 ammonia
Base acid sequence (when interest recombinant protein is GrM), SEQ ID NO：73 or 75 (when interest recombinant protein is IFN α), SEQ
ID NO：78 or 80 or 82 (when interest recombinant protein is cancer antigen SSX2), SEQ ID NO：85 or 87 (when interest recombinates egg
When being NERCMSL in vain), SEQ ID NO：90 (when interest recombinant protein is FasL) or SEQ ID NO：93 (when interest restructuring
When albumen is CNTN4).
Do not degraded it is interesting that the fusion protein of the present invention can be stored at 4 DEG C lasting some months.During storage
This stabilization in vitro effect can be attributed to mgmt protein support effect, and/or obtained due to the presence of mgmt protein
High concentration (typically at least 40mg/mL).
Importantly, and MGMT combination stabilize recombinant protein in the purge process of secretory protein.Therefore once
It is applied in the subject of this demand, it can be used for stablizing recombinant protein in vivo.It will be for strengthening this with MGMT couplings
The means in kind of recombinant protein life-span in vivo.During this internal stablizing effect is currently studied.
In the 5th aspect, the present invention relates to the spinal animal recombinant host cell including expression vector of the present invention.
Spinal zooblast can come from insect, Arachnoidea, Crustachia, Mollusca, Annelida, cirrus
Guiding principle, Radiata, any cell of Coelenterata and Ciliata.In the context of the present invention, spinal zooblast
Preferably insect cell, such as drosophila or mosquito cells.They are more preferably Drosophila S 2 cells.
Drosophila S 2 cells have been widely described.They are especially suitable for the high yield production of albumen, because in room temperature (24 ± 1
DEG C) under they may remain in suspension culture in.Culture medium is the heat inactivation tire ox blood being supplemented between 5% and 10% (v/v)
The M of (FBS) clearly3.In a preferred embodiment of the invention, culture medium contains 5%FBS.After induction, cell culture is in serum-free
In medium.In such medium, S2 cells can grow in the culture that suspends, such as in 250mL into 2000mL revolving bottles,
Stirred with 50-60rpm.Cell density generally remains in 106With 107Between cell per mL.
The invention further relates to the restructuring S2 drosophila cells including expression vector of the present invention, the expression vector includes preferred choosing
From following nucleotide sequence：
- plasmid SEQ ID NO：64 (pUC57 for carrying DeSNAP Univ) or the nucleotide sequence being cloned into cell,
According to budapest treaty, the cell has been deposited in French national microorganism on December 9th, 2011 with numbering CNCM I-4581
Collection (CNCM), Institute Pasteur, Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux
No. 25-28,
- include SEQ ID NO：19 carrier or the nucleotide sequence being cloned into cell, should according to budapest treaty
Cell is deposited in French national Organism Depositary (CNCM), Bath in August in 2010 19 days with numbering CNCM I-4357
Moral research institute, Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28,
- include SEQ ID NO：22 carrier of the present invention or the nucleotide sequence being cloned into cell, the cell in
On October 27th, 2010 is deposited in French national Organism Depositary (CNCM), Pasteur research with numbering CNCM I-4381
Institute,
- include SEQ ID NO：21 carrier of the present invention or the nucleotide sequence being cloned into cell, the cell in
On October 27th, 2010 is deposited in French national Organism Depositary (CNCM), Pasteur research with numbering CNCM I-4382
Institute,
-SEQ ID NO：9 carrier or the nucleotide sequence being cloned into cell, the cell is in September in 2010 29 days
French national Organism Depositary (CNCM) is deposited in numbering CNCM I-4368, Institute Pasteur,
- include SEQ ID NO：20 carrier of the present invention or the nucleotide sequence being cloned into cell, the cell in
September is deposited in French national Organism Depositary (CNCM), Pasteur research on 29th with numbering CNCM I-4369 within 2010
Institute,
-SEQ ID NO：71 carrier,
- include SEQ ID NO：57 or 72 or 74 (when protein of interest is IFN α), SEQ ID NO：77th, 79 or 81 (when
When protein of interest is cancer antigen SSX2), SEQ ID NO：55 (when protein of interest is granzyme M), SEQ ID NO：89 (when emerging
When interesting albumen is FasL), SEQ ID NO：84 or 86 (when protein of interest is cancer antigen NERCMSL) or SEQ ID NO：92
(when protein of interest is contactin CNTN4) or SEQ ID NO：This hair of 96 (when protein of interest is hSULF-2 Δ TMD)
Bright carrier.
The S2 cells of stable transfection of the present invention are also selected from as follows：
- French national Organism Depositary (CNCM) is deposited in numbering CNCM I-4357 in August in 2010 19 days,
The cell of Institute Pasteur (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
(S2/SNAP+RVF-N),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4381 on October 27th, 2010
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+WN.EDIII*),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4382 on October 27th, 2010
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+IFNAI*),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4368 in September in 2010 29 days
Cell (the pDe of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
Snap-1),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4369 in September in 2010 29 days
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
WNsE+SNAP),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4565 on December 5th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+DEN1.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4566 on December 5th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+DEN2.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4567 on December 5th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+DEN3.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4568 on December 5th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+DEN4.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4569 on December 5th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+YF.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4570 on December 5th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+JE.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4571 on December 5th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+USU.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4572 on December 5th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+TBE.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4576 on December 8th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+MVE.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4577 on December 8th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+Rocio.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4578 on December 8th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+SLE.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4579 on December 8th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+WSL.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4580 on December 8th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP+Zika.EDIII),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4581 on December 9th, 2011
The cell of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
(pDeSnapUniv),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4582 on December 9th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP-MUB70),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4583 on December 9th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP-SSX2),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4584 on December 9th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAPuniv-NERCMSL),
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4585 on December 9th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP-huGrM), and
- French national Organism Depositary, Pasteur be deposited in numbering CNCM I-4586 on December 9th, 2011
Cell (the S2/ of research institute (Paris, FRA, postcode 75724, mailbox 15, rue du Docteur Roux 25-28)
SNAP-proTEV)。
Recombinant cell with numbering CNCM I-4357 preservations is to include SEQ ID NO：19 plasmid vector (pMT/BiP/
SNAP-RVF.N/His labels) stable macrophage drosophila cell system S2, wherein RVF.N for Rift Valley fever virus (RVF) N resist
Original (see Brehin et al., Virology 371:185,2008).
Recombinant cell with numbering CNCM I-4381 preservations is to include the stabilization of plasmid vector pMT/BiP/V5-His labels
Macrophage drosophila cell system S2, wherein SEQ ID NO：22 (SNAP/WN.EDIII) are inserted into after BiP sequences, wherein
WN.EDIII is the III domains of west nile virus glycoprotein E.
Recombinant cell with numbering CNCM I-4382 preservations is to include the stable macrophage of plasmid vector pMT/V5-His labels
Cell drosophila cell system S2, wherein inserted with SEQ ID NO：21(BiP/SNAP/IFNα1).IFN α 1 is SEQ ID NO：32
The interferon of people α 1 (Mokkim et al., Protein expression purif.63:140,2009).
Recombinant cell with CNCM I-4369 preservations is included containing SEQ ID NO：20 (WN.sE/SNAP/his labels)
Plasmid vector pMT/BiP/V5-His labels stable macrophage drosophila cell system S2, wherein WN.sE is west nile virus E
The soluble form of envelope protein.
Recombinant cell with CNCM I-4369 preservations is included containing SEQ ID NO：20 (WN.sE/SNAP/his labels)
Plasmid vector pMT/BiP/V5-His labels stable macrophage drosophila cell system S2, wherein WN.sE is west nile virus E
The soluble form of envelope protein.
Recombinant cell with CNCM I-4565 preservations is to include plasmid vector pMT/BiP/SNAP+DV1.EDIII/His marks
The stable macrophage drosophila cell system S2 of label, wherein the EDIII albumen of DV1.EDIII encoding dengues virus 1, and with sequence
SEQ ID NO：27.
Recombinant cell with CNCM I-4566 preservations is to include plasmid vector pMT/BiP/SNAP+DV2.EDIII/His marks
The stable macrophage drosophila cell system S2 of label, wherein the EDIII albumen of DV2.EDIII encoding dengues virus 2, and with sequence
SEQ ID NO：28.
Recombinant cell with CNCM I-4567 preservations is to include plasmid vector pMT/BiP/SNAP+DV3.EDIII/His marks
The stable macrophage drosophila cell system S2 of label, wherein the EDIII albumen of DV3.EDIII encoding dengues virus 3, and with sequence
SEQ ID NO：29.
Recombinant cell with CNCM I-4568 preservations is to include plasmid vector pMT/BiP/SNAP+DV4.EDIII/His marks
The stable macrophage drosophila cell system S2 of label, wherein the EDIII albumen of DV4.EDIII encoding dengues virus-4, and with sequence
SEQ ID NO：30.
Recombinant cell with CNCM I-4569 preservations is to include plasmid vector pMT/BiP/SNAP+YF.EDIII/His marks
The stable macrophage drosophila cell system S2 of label, wherein YF.EDIII encode the EDIII albumen of yellow fever virus, and with sequence
SEQ ID NO：31.
Recombinant cell with CNCM I-4570 preservations is to include plasmid vector pMT/BiP/SNAP+JE.EDIII/His marks
The stable macrophage drosophila cell system S2 of label, wherein JE.EDIII encode the EDIII albumen of japanese encephalitis virus, and with sequence
Arrange SEQ ID NO：25.
Recombinant cell with CNCM I-4571 preservations is to include plasmid vector pMT/BiP/SNAP+USU.EDIII/His marks
The EDIII albumen of the stable macrophage drosophila cell system S2 of label, wherein USU.EDIII coding Usu figure virus, and with sequence
Arrange SEQ ID NO：24.
Recombinant cell with CNCM I-4572 preservations is to include plasmid vector pMT/BiP/SNAP+TBE.EDIII/His marks
The stable macrophage drosophila cell system S2 of label, wherein TBE.EDIII encode the EDIII albumen of Far East Russian encephalitis virus, and have
There are sequence SEQ ID NO：26.
Recombinant cell with CNCM I-4576 preservations is to include plasmid vector pMT/BiP/SNAP+MVE.EDIII/His marks
The stable macrophage drosophila cell system S2 of label, wherein MVE.EDIII encode the EDIII albumen of Murray encephalitis viruses.
Recombinant cell with CNCM I-4577 preservations is to include plasmid vector pMT/BiP/SNAP+Rocio.EDIII/His
The stable macrophage drosophila cell system S2 of label, wherein Rocio.EDIII encode the EDIII albumen of rocio virus.
Recombinant cell with CNCM I-4578 preservations is to include plasmid vector pMT/BiP/SNAP+SLE.EDIII/His marks
The stable macrophage drosophila cell system S2 of label, wherein SLE.EDIII coding St. Louis encephalitis virus (Saint-Louis
Encephalitis virus) EDIII albumen.
Recombinant cell with CNCM I-4579 preservations is to include plasmid vector pMT/BiP/SNAP+WSL.EDIII/His marks
The EDIII albumen of the stable macrophage drosophila cell system S2 of label, wherein WSL.EDIII coding Wei Saiersi Blang's viruses.
Recombinant cell with CNCM I-4580 preservations is to include plasmid vector pMT/BiP/SNAP+Zika.EDIII/His
The stable macrophage drosophila cell system S2 of label, wherein Zika.EDIII coding hereby block the EDIII albumen of virus.
Plasmid vector pMT/BiP/SNAP+SSX2/His labels are included with the recombinant cell of CNCM I-4583 preservations
Stable macrophage drosophila cell system S2, wherein SSX2 are SEQ ID NO：76.
Recombinant cell with CNCM I-4584 preservations is to include plasmid vector pMT/BiP/SNAP+NERCMSL/His labels
Stable macrophage drosophila cell system S2, wherein NERCMSL is SEQ ID NO：83.
Recombinant cell with CNCM I-4585 preservations is include plasmid vector pMT/BiP/SNAP+GrM/His labels steady
It is SEQ ID NO to determine macrophage drosophila cell system S2, wherein GrM：54.
Recombinant cell with CNCM I-4586 preservations is to include the stabilization of plasmid vector pMT/BiP/ProTEV/His labels
Macrophage drosophila cell system S2, wherein ProTEV are SEQ ID NO：52.
In the 6th aspect, present invention is alternatively directed to the vertebrate recombinant cell that stable transfection has expression vector of the present invention.
Preferably, the vertebrate recombinant cell is mammalian cell, preferably CHO, YB2/O, COS, HEK,
NIH3T3, HeLa cell or derivatives thereof.It is further preferred that in this case, expression vector of the invention includes SEQ ID NO：
57 or 72 or 74 (when protein of interest is IFN α), SEQ ID NO：77th, 79 or 81 (when protein of interest is cancer antigen SSX2),
SEQ ID NO：55 (when protein of interest is granzyme M), SEQ ID NO：89 (when protein of interest is FasL), SEQ ID
NO：84 or 86 (when protein of interest is cancer antigen NERCMSL), SEQ ID NO：92 (when protein of interest is contactin CNTN4
When) or SEQ ID NO：96 (when protein of interest is hSULF-2ΔTMDWhen).In the 7th aspect, egg is recombinated the present invention relates to enhancing
The method expressed in vain, including by the albumen and peptide secretion signal and enzyme 6- methyl guanine-DNA- transmethylases (MGMT)
(EC 2.1.1.63), its mutant or catalyst structure domain coexpression.The coexpression is preferably entered in spinal zooblast
OK, and more preferably insect cell.
It is further preferred that in the method, MGMT enzymes are SEQ ID NO：4 albumen or its homologue, such as SEQ ID NO：2
SNAP albumen or its homologue.
In the context of the present invention, " Enhanced expressing " of term heterologous protein refers to the albumen in recombinant cell supernatant
In or cell itself in expression, compared to the recombinant vector of prior art, (that is, be not co-expressed albumen and MGMT or SNAP albumen
Carrier) expression and/or secretion of the albumen that are obtained, improve at least 2 times, preferably 5 times, more preferably 10 times, and even
More preferably 20 times.In one preferred embodiment, term " Enhanced expressing " refers to be possible to have carrier of the present invention from transfection
Host cell supernatant in reclaim at least 40mg/L, preferably at least 50mg/L, more preferably at least 60mg/L protein of interest.
Term " coexpression " refers to coding i) recombinant protein, ii) MGMT enzymes, its mutant or catalyst structure domain and iii) peptide
The DNA sequence dna of secretion signal, is operably coupled to expression regulation sequence (i.e. transcription and translation regulating and controlling sequence), and controlled by it
System.Therefore the translation of the DNA sequence dna of encoded peptide secretion signal, interest heterologous protein and MGMT enzymes causes the formation of fused polypeptide,
Wherein albumen can be separated by intervening sequence as defined above, and/or cleavage sites.
" the peptide secretion signal " of fused polypeptide of the present invention preferably in spinal zooblast or vertebrate cells or
Has functional secretion signal in both and more preferably in insect cell, even more preferably in Drosophila S 2 cells.
The example of tool functional peptide secretion signal is in insect cell：SEQ ID NO：48 insect ssBiP, SEQ
ID NO：Any peptide signal present in 51 BiP samples signal and arboviruse, the envelope E protein of such as west nile virus
(SEQ ID NO：15).
The example for having functional peptide secretion signal in vertebrate and spinal zooblast is SEQ ID NO：51
BiP sample signals.
In eighth aspect, restructuring egg is produced the present invention relates to the production for improving interest recombinant protein or in cell culture
White method, including the use of carrier of the invention as described above or recombinant host cell as described above.
More accurately, improve the production of interest recombinant protein or the methods described of recombinant protein is produced in cell culture
Including step：
A) polynucleotide expression vector of the invention of the coding protein of interest is provided,
B) expression vector is introduced into host cell, preferably spinal animal or vertebrate host cell,
C) nucleotides of the introducing host cell is made to be expressed to produce the interest recombinant protein.
Preferably, the spinal animal host cell is insect cell, such as Drosophila S 2 cells.
Preferably, the vertebrate host cell is mammalian cell, such as CHO, YB2/O, COS, HEK,
NIH3T3, HeLa cell or derivatives thereof.
By using this method, interest recombinant protein is obtained with the cells and supernatant of at least 40mg/L or more recovery
To express.
The use of the drosophila cell system S2 that gene outcome is directly secreted into medium is the side of being preferable to carry out of the present invention
Formula (being directly secreted into medium allows to utilize efficient step purification system).
In the 9th embodiment, the present invention relates to enzyme 6- methyl guanine-DNA- transmethylases (MGMT) (EC
2.1.1.63), its mutant or catalyst structure domain are used to strengthen recombinant protein preferably infected with science or defective vector
Spinal animal and/or vertebrate host cell, the more preferably level of production in insect cell or mammalian cell
Purposes.
MGMT enzymes can be sequence SEQ ID NO：4 people MGMT (referring to NP_002403.2), it is accredited as NP_
032624.1 mouse MGMT (SEQ ID NO：45) NP_036993.1 rat MGMT (SEQ ID NO, are accredited as：46), its
Homologous sequence or its sub-piece.
Preferably, MGMT mutant enzymes are SEQ ID NO：2 SNAP albumen or its homologue, i.e., with sequence SEQ ID
NO：2 SNAP albumen at least more than 80%, preferably 85%, more preferably 90% homogeneity.
The spinal zooblast is preferably insect cell, such as Drosophila S 2 cells.
In one preferred embodiment, the present invention relates to enzyme 6- methyl guanine-DNA- transmethylases (MGMT)
(EC 2.1.1.63), its mutant or catalyst structure domain are used to strengthen recombinant protein infected with science or defective vector
Vertebrate cells, such as the purposes of the level of production in mammalian cell.
The vertebrate cells are preferably EBX, CHO, YB2/O, COS, HEK, NIH3T3 cell or derivatives thereof.
In addition, being used to improve interest egg the present invention relates to the DNA sequence dna of coding MGMT enzymes, its mutant or catalyst structure domain
The purposes of the white level of production in recombinant cell.
It is used for i) in vitro and in vivo the invention further relates to the DNA sequence dna for encoding MGMT enzymes, its mutant or catalyst structure domain
Stabilized interest recombinant protein, and therefore ii) strengthen the purposes in its in vitro and in vivo life-span.
Such as this DNA sequence dna is people's mgmt gene sequence NM_002412.3, (the SEQ ID NO of gene I/D 4255：3) or
Its any homologous sequence (preferably SEQ ID NO of encoding function MGMT enzymes, its mutant or catalyst structure domain：1、SEQ NO：
47、SEQ ID NO：67 or SEQ ID NO：68).
Particularly, the present invention relates to 6- methyl guanine-DNA- transmethylases (MGMT, EC 2.1.1.63), its mutation
Body or catalyst structure domain, as the protection polypeptide for being fused to or being connected to recombinant protein, come improve recombinant protein storage medium,
The purposes of half-life period in blood plasma or buffer solution, to improve the purposes of the recombinant protein half-life period as medicine or vaccine, or changes
It is apt to the purposes for the recombinant protein half-life period in diagnostic kit.
In the context of the present invention, " the improvement level of production " or " the enhancing level of production " of term heterologous protein refers to institute
Albumen is stated in the cell conditioned medium or intracellular expression, compared to the recombinant vector of prior art (that is, not including this hair
Bright carrier) expression of the albumen that is obtained, improve at least 2 times, more preferably preferably 5 times, 10 times, even more preferably 20
Times.In one preferred embodiment, term " improving production " refers to be possible to have the host of carrier of the present invention thin from transfection
At least 40mg/L, preferably at least 50mg/L, more preferably at least 60mg/L protein of interest is reclaimed in born of the same parents' supernatant.
In one preferred embodiment, the recombinant protein is selected from：Insulin, IFN, SSX2, granzyme M, FasL,
Mesotheline (NERMCSL), interior sulfatase (hSULF) or contactin.
In a specific embodiment, the invention further relates to the method for producing interest recombinant protein, method bag
Include step：
(a) allogeneic dna sequence of coding interest recombinant protein is obtained；
(b) allogeneic dna sequence is inserted to the polynucleotide expression vector of the present invention, the carrier has such as DNA sequences
Arrange SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：64 or SEQ ID NO：71,
(c) the polynucleotides transfection host cell (preferably insect cell or mammalian cell) obtained with step (b)；
(d) polynucleotides for obtaining step (c) are expressed to produce protein of interest；
(e) optionally, MGMT polypeptides are cut,
(f) protein of interest is reclaimed,
(g) optionally, protein of interest is purified.
, can be using the conventional molecular biology in the range of art technology in order to carry out the different step of the inventive method
, microbiology and recombinant DNA technology.This technology has complete explanation in the literature.See, for example, Sambrook,
Fitsch&Maniatis,Molecular Cloning:A Laboratory Manual, the second edition (1989) Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein with reference to " Sambrook et al., 1989 ")；
DNA Cloning:A Practical Approach, Volumes I and II (D.N.Glover edits 1985)；
Oligonucleotide Synthesis (M.J.Gait edits 1984)；Nucleic Acid Hybridization
(B.D.Hames&S.J.Higgins,eds.1984)；(R.I.Freshney, editor is 1986) by Animal Cell Culture；
Immobilized Cells and Enzymes(IRL Press,1986)；B.E.Perbal,A Practical Guide to
Molecular Cloning(1984)；F.M.Ausubel et al., (editor), Current Protocols in Molecular
Biology,John Wiley&Sons,Inc.(1994)。
Term " transfection " refers to exogenous nucleic acid being incorporated into eukaryotic host cell, so that host cell will express introduced
Gene or sequence are introduced gene or the albumen of sequential coding to produce required material, in the present invention.Receive and express
Introduced DNA or RNA host cell are " being transfected " and turn into " transfectant " or " clone ".It is incorporated into host cell
DNA or RNA can come from any source, including belong to host cell the cell of same genus or kind, or belong to and differ category
Or the cell planted.
In the context of the present invention, it can be entered with polynucleotides transfection host cell by the classical way in this area
OK, transfection, infection or electroporation are for example passed through.In another embodiment, can be by liposome transfection (with naked DNA
Form), or with other transfections (peptide, polymer etc.) in vivo introduce the present invention carrier.The cation lipid of synthesis
Available for the internal transfection for the gene for preparing coding maker thing liposome (Felgner et al.,
Proc.Natl.Acad.Sci.U.S.A.,84:7413-7417,1987).Lipid compounds or combination available for transfer nucleic acid
Thing is described in WO 95/18863 and WO 96/17823, and U.S.5,459,127.For the purpose of targeting, lipid is chemically
Other molecules are coupled to (see Mackey et al., Proc.Natl.Acad.Sci.U.S.A., 85:8027-8031,1988).Targeting
Peptide such as hormone or neurotransmitter and albumen such as antibody or non-peptide molecule can be with chemical couplings to liposome.Other molecules also can use
In promoting the transfection of nucleic acid in vivo, such as cation oligopeptides (referring to WO95/21931), the peptide from DBP are (see WO
Or cationic polymer 96/25508) (see WO 95/21931).May also in vivo it draw carrier as naked DNA plasmid
Enter.Such as electroporation, microinjection, cell fusion, deae dextran, calcium phosphate precipitation, it can be used by means known in the art
Particle gun uses DNA vector transporter, (see Wu in host cell needed for the naked DNA carrier for gene therapy is introduced into
Et al., J.Biol.Chem., 267:963-967,1992；Wu and Wu, J.Biol.Chem., 263:14621-14624,1988；
Williams et al., Proc.Natl.Acad.Sci.U.S.A., 88:2726-2730,1991).
Term " allowing expression ", polynucleotides were referred to herein as the regulating and controlling sequence and all required compositions being present in carrier
Stimulate (such as activation-inducing type promoter), it is carried out with being enough the amount for occurring polynucleotides translation.
If desired, the MGMT/SNAP polypeptides of produced fusion protein cut through into supernatant or recombinated
It is intracellular to add the protease with clear and definite cleavage site and realize.For example, by adding enterokinase into recombinant cell supernatant
And obtain enterokinase cleavage site point DDDK/D cutting.Or, MGMT/SNAP polypeptides can be maintained to strengthen recombinant protein
Life-span.
In addition, technical staff will be understood that can be detected in the culture medium for maintaining or cultivating this host cell expression or
The albumen or polypeptide of secretion.By known method, such as centrifuge or filtering can separate culture medium and host cell.Then, lead to
The known properties feature using albumen or polypeptide is crossed, albumen or polypeptide can be detected in cell-free medium.This property can
So that the difference including albumen or polypeptide is immune, enzyme or physical property.For example, if albumen or polypeptide have unique enzymatic activity,
Can be in the enterprising hand-manipulating of needle of host cell used medium to this active analysis.In addition, being directed to given albumen when that can obtain
Or polypeptide reactive antibody when, this antibody can be used for detecting in any known immunological method albumen or polypeptide (for example,
In Harlowe, et al., 1988, Antibodies:A Laboratory Manual,Cold Spring Harbor
In Laboratory Press).
The recovery of protein of interest is mediated by method as known in the art, including but not limited to preparative disc gel electricity
Swimming, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and salting-out chromatdgraphy, extraction and inverse
Stream distribution (countercurrent distribution) etc..Because it is preferred that in markd recombination system of the present invention is connected
Protein of interest is produced, the mark contributes to the recovery from host cell thick lysate on appropriate solid-phase matrix by chromatogram
Polypeptide.Or, the antibody of the polypeptide of confrontation albumen or confrontation from albumen of generation can be used as recovery catalyst.
Can implement further purification step (g), but what is interesting is and need not.
The material being purified can contain less than about 50%, preferably less than about 75%, most preferably less than about 90% and the quilt
The original related cell component of material of purifying.It is " substantially pure " that represent can be with using conventional purification technique known in the art
The highest purity of realization.
In an embodiment of the invention, method of the invention allows to obtain in the cells and supernatant of recovery
At least 40mg/L, preferably at least 50mg/L, more preferably at least 60mg/L substantially pure protein of interest.
(i.e. coupling has the recombinant protein of MGMT/SNAP polypeptides to the interest recombinant protein and fusion protein of the present invention, and it is than list
Only recombinant protein is more stable) it can be used in various products.For example, these restructuring and/or fusion protein can be used for drug regimen
Thing, such as treatment for virus infection.
In one preferred embodiment, the recombinant protein is selected from：Insulin, IFN, FasL, granzyme M, SSX2,
Mesotheline (NERMCSL), interior sulfatase (hSULF) or contactin.
In another embodiment, the present invention provides the infectious viral particle containing above-mentioned nucleic acid carrier.Usual feelings
Under condition, such virion is produced by the method comprised the following steps：
(a) viral vector of the present invention is incorporated into suitable cell line,
(b) cell line is cultivated under suitable condition to allow to produce the infectious viral particle,
(c) produced infectious viral particle is reclaimed from the culture of the cell line, and
(d) infectious viral particle of the recovery is optionally purified.
When viral vector is deficiency, generally it is trans offer non-functional viral gene complementary cell system or pass through
Infectious particles are produced using helper virus.For example, the appropriate cell line for the complementary E1 adenovirus vectors lacked includes
293 cells and PER-C6 cells.Infectious viral particle can be reclaimed from culture supernatant or from the cell after cracking.Root
According to standard technique (for example WO96/27677, WO98/00524, WO98/22588, WO98/26048, WO00/40702,
Chromatography described in EP1016700 and WO00/50573, the supercentrifugation in cesium chloride gradient) they can be entered one
Step purifying.
Therefore, the present invention relates to expression vector, recombinant protein, fusion protein, host cell or the virus for including the present invention
The pharmaceutical composition of particle or its any combination.Such pharmaceutical composition include be mixed with pharmaceutical acceptable carrier, by this hair
Carrier, particle, cell or the albumen for the therapeutic dose that bright method is obtained.
Parenteral, vein or hypodermically systemic administration composition can be passed through.When systemic administration, for the present invention
Therapeutic combination for without pyrogen, the acceptable protein solution form of parenteral.This acceptable albumen of parenteral is molten
The preparation of liquid, it is contemplated that pH, isotonicity, stability etc., within the skill of the art.
In view of changing pharmaceutically-active many factors, such as state, body weight, the excreta (sew) of patient and diet, sense
The order of severity, time of application and the other clinical factors of dye, dosage will be determined by attending doctor.The medicine of pharmaceutical composition
Carrier and other compositions will be selected by those skilled in the art.
In addition, for example, the present invention fusion and recombinant protein can be used as vaccine composition, come seeded with mammalian subject with
To viral infection resisting.These albumen can be used alone or are used in combination with other recombinant proteins or Therapeutic Vaccination agent.This epidemic disease
The composition of seedling will be determined by those skilled in the art.
Present invention additionally comprises fusion protein, expression vector, infectious viral particle, host cell or the medicine group of the present invention
Compound is used to prepare the purposes in medicine particularly vaccine.
The present invention also provides the method for treating or preventing human or animal organism, including is controlled to organism administration
Treat fusion protein of the invention, expression vector, infectious viral particle, host cell or the composition of effective dose.
The SNAP albumen of the albumen, especially interest fusion protein of the last present invention, can be used as being used to detect cancer, virus
The diagnosticum that antiviral protein antibody is present in infection or biological fluid (such as blood, serum, saliva).These albumen also can use
The method of virus protein in identification and/or isolated organism liquid and tissue.Therefore, albumen can be the examination for carrying out this method
Composition in agent box.
Therefore, in another aspect, the invention further relates to by any method of the invention obtain from pathogenic or
The recombinant protein of non-pathogenic microorganisms or the recombinant protein of MGMT or SNAP labels, which are used to identify in biological specimen, whether there is
Described pathogenic or non-pathogenic microorganisms purposes.In one preferred embodiment, the pathogenic microorganisms is virus,
The albumen of MGMT or SNAP labels is virus protein, such as comes from chikungunya, Dengue, encephalitis B (JE), tick-borne encephalitis
(TBE), the EDIII of yellow hot (YF), Usu figure (USU) or west nile virus or the core from Rift Valley fever or toscana virus
Albumen N.
In the context of the present invention, the biological specimen refers to blood sample, urine sample or the doubtful any life being infected
Thing sample.
Embodiment
1. plasmid construction
1.1. plasmid pMT/BiP/V5-His A are used.It includes 3642 nucleotides, and includes following characteristics：
- metallothionein promoter：Base 412-778
- transcription initiation：Base 778
- MT forward primers site：Base 814-831
- BiP signal sequences：Base 851-904 (SEQ ID NO：11)
- multiple cloning sites：Base 906-999
- V5 antigenic tags：Base 1003-1044
- polyhistidine area：Base 1054-1074
- BGH reverse primers site：Base 1094-1111
- SV40 late polyadenylation signals：Base 1267-1272
- pUC origins：Base 1765-2438 (complementary strand)
- bla promoters：Base 3444-3542 (complementary strand)
- ampicillin (bla) resistant gene ORF：Base 2583-3443 (complementary strand)
PUC57Amp carriers can be used for the purpose of the present invention.The carrier is included：
- unique cloning site EcoRI
- metallothionein promoter,
- geneome RNA 5' ends the noncoding region from west nile virus strain IS-98-ST1,
- translation initiation codon (ATG),
Signal peptide (the SEQ ID NO of-envelope E protein from west nile virus strain IS-98-ST1：15)、
- from west nile virus strain IS-98-ST1 geneome RNA 3' ends noncoding region, two of which repetitive sequence and
3' ends stem ring be deleted,
- S40polyA signals motif,
- unique cloning site ApaI.
1.2.SNAP clone
On template pMT/BiP/CHIK.sE2+SNAP labels, a pair of 5'-SNAP as described below are used by PCR
Coding SNAP protein sequence SEQ ID NO are carried out with 3'-MCS primers：2 DNA amplification.
Primer 5'-SNAP：5’-aaaaaagatctgacaaagactgcgaaatg-3’(SEQ ID NO：7)
Primer 3'-MCS：5’-gaggagagggttagggataggcttacc-3’(SEQ ID NO：8)
Then with Bgl II and Not I digestion PCR primers, and it is inserted into DES system neutral plasmids p/MT/BiP/
Between V5-A unique Bgl II (at MCS 5' ends) and Not I (at MCS 3' ends) site.
Obtained plasmid is SEQ ID NO：9 pMT/BiP/SNAP-His label carriers, including：
-SEQ ID NO：11 insect ssBiP sequences,
-SEQ ID NO：1 SNAP DNA sequence dnas,
-SEQ ID NO：12 enterokinase cleavage site point,
- EcoRV-Sma I restriction sites,
- coding the His positioned at restriction site downstream6DNA (the SEQ ID NO of label：14) and
- be located at i) between enhancer sequence and EcoRV-SmaI restriction sites, and ii) EcoRV-SmaI restriction sites
With coding His6SEQ ID NO between the DNA of label：13 two DNA intervening sequences.
PMT/BiP/SNAP-His label carriers can be obtained from pUC57 skeletons, and obtain with sequence SEQ ID NO：10
Carrier, carrier includes：
- unique cloning site EcoRI
- metallothionein promoter
- 5' ends the noncoding region from west nile virus strain IS-98-ST1 geneome RNAs,
- translation initiation codon,
-SEQ ID NO：The signal peptide of 15 envelope E protein from west nile virus strain IS-98-ST1,
-SEQ ID NO：47 SNAP DNA sequence dnas,
- be used for by exogenous array insert framework in unique cloning site EcoR V and Sma I/Xma I,
-SEQ ID NO：12 enterokinase cleavage site point, its be located between SNAP enhancer DNAs and cloning site,
DNA (the SEQ ID NO of six His-Tag sequences of-coding：14)、
- be located at i) between enhancer sequence and EcoRV-SmaI restriction sites, and ii) EcoRV-SmaI restriction sites
With coding His6SEQ ID NO between the DNA of label：13 two DNA intervening sequences
- two translation termination codons,
- from west nile virus strain IS-98-ST1 geneome RNAs 3' ends noncoding region, two of which repetitive sequence and
3' ends stem ring be deleted,
- S40polyA signals motif and
- unique cloning site ApaI.
PMT/BiP samples/SNAP-His label carriers can be obtained from pUC57 skeletons, wherein SEQ ID NO：59 (also see Fig. 8 A
And such as 8B) be inserted between unique site EcoR V and Hind III.Carrier has sequence SEQ ID NO：64.It includes：
- unique cloning site EcoRI
- metallothionein promoter
- 5' ends the noncoding region from west nile virus strain IS-98-ST1 geneome RNAs,
- translation initiation codon,
-SEQ ID NO：The signal peptide of 15 envelope E protein from west nile virus strain IS-98-ST1,
-SEQ ID NO：47 SNAP DNA sequence dnas,
- be used for by exogenous array insert framework in unique cloning site Bam H1, EcoR V, Apa I and Xma I,
-SEQ ID NO：52 two proTEV cleavage sites, between SNAP enhancer DNAs and His labels,
DNA (the SEQ ID NO of six His-Tag sequences of-coding：14)、
- be located at i) between enhancer sequence and EcoRV-SmaI restriction sites, and ii) Apa I restriction sites and volume
Code His6SEQ ID NO between the DNA of label：13 two DNA intervening sequences
- two translation termination codons,
- from west nile virus strain IS-98-ST1 geneome RNAs 3' ends noncoding region, two of which repetitive sequence and
3' ends stem ring be deleted,
- S40polyA signals motif and
- unique cloning site ApaI.
1.3. the clone of interest genes
1.3.1. the nucleoprotein N of Rift Valley fever virus (RVF-N)
Pass through PCR determining using the cDNA of 5'-N and 3'-N' primer pairs progress coding RFV-N protein sequences listed below
To mutagenesis.
Primer 5'-N：
5'-aaaaaggcgcgccagggggtggcggatctgacaactatcaagagcttcgagtccagtttgctgctc
-3'(SEQ ID NO：17)
Primer 3'-N：
5'-aaaaaaccggtcaatgatgatgatgatgatgacttccaccgccggctgctgtcttgtaagcctgag
cgg-3'
(SEQ ID NO：18)
1.3.2. non-viral albumen, such as interferon IFN α 1 or granzyme M
SEQ ID NO can also be used：32 protein sequence of people's IFN α 1.
SEQ ID NO can also be used：54 human granular enzyme M protein sequences.Granzyme M is chymotrypsin-like serine albumen
Enzyme, it preferentially cuts its substrate after Met or Leu.Its constitutive expression in the intrinsic effect NK of activation.
This protease also has antiviral and antitumor properties (van Domselaar R. et al., The Journal of
Immunology 2010；Hu D. et al., The Journal of Biological Chemistry 2010).
1.4. the gene of encoding proteins is inserted into pMT/BiP/SNAP-His labels or pMT/BiP samples/SNAP-His is marked
Carrier is signed, so as to obtain pMT/BiP/SNAP- albumen-His labels or pMT/BiP samples/SNAP- albumen-His label carriers
1.4.1.RVF.N
The PCR primer obtained in 1.3.1 is digested by BssHII and AgeI, and is inserted into the linearisation obtained in 1.2
The unique BssHII (at the 3' ends of SNAP genes) and AgeI of plasmid p/MT/BiP/SNAP-His labels are (shuttle plasmid MCS's
3' ends) between site.
Obtained plasmid is such as p/MT/BiP/SNAP-RVF.N/His labels (SEQ ID NO：19).
1.4.2. the ED III albumen of different flavivirus
In order to strengthen the specificity of ELISA or western blot test based on recombinant flavivirus antigen, E protein (ED III)
Antigenic domains III seemingly plant promising method (Ludolfs et al., 2007).Come from the method test of the present invention
Xi Niluo (WN), Usu figure (USU), encephalitis B (JE), tick propagate encephalitis (TBE), dengue serotypes 1 to 4 (DEN-1, -2, -
3rd, -4) and Huang hot (YF) virus restructuring EDIII production.
WN, USU and JE virus of zoonosis belong to JE serum complexes (serocomplex).Drosophila S2 induction types
Expression system (DES, Invitrogen) has been selected for mass producing from flavivirus in spinal zooblast
Individual EDIII.Encode the E protein total length Domain III from flavivirus WN, USU, JE, TBE, DEN-1 to DEN-4 and YF
Synthetic gene is listed in SEQ ID NO：23 to SEQ ID NO：31.In the 1.2 plasmid pMT/BiP/SNAP-His labels obtained,
The C-terminal that ED III sequences are fused to SNAP albumen in the frame produces fusion protein S NAP-EDIII.
1.4.3.IFNα
Obtain plasmid pMT/BiP/SNAP-IFN-His labels (see Fig. 4 A to Fig. 4 F) and pMT/BiP samples/SNAP-IFN-
His labels (refer to Fig. 8 A and Fig. 8 B).
1.4.4. granzyme M
Obtain plasmid pMT/BiP/SNAP-GrM-His labels (referring to Fig. 6 A to 6C).
2. it is transfected into host cell
2.1. it is transfected into S2 cells
Allow SNAP- label proteins as with HisLabelThe gained plasmid that the secretion fusion protein of ending is produced
PMT/BiP/SNAP- albumen-HisLabelDisplay ammonia benzyl mould is generated into S2 cells with selection marker thing pCO-Blast cotransfections
The stable S2/sSNAP- albumen-His of plain resistanceLabelCell line.
The stable S2 cell lines of growth are stimulated 10 days with heavy metal cadmium (Cd2+) in blender jar (spinner) (1000ml),
Concentrate and purify the albumen from extracellular medium.
After being incubated 10 days with heavy metal cadmium, observe and divide in the cell conditioned medium for stablizing S2/sSNAP- albumen-His labels
The accumulation for the SNAP- label proteins secreted.
With the sheep blood serum of anti-His labels, immunoblotting assay make it possible to detect extracellular SNAP- label proteins (see
Fig. 2 B, 3B, 4B, 6C).
2.2. it is transfected into HeLa cells
Plasmid pMT/BiP samples/SNAP-IFN-HisLabelIt is transfected into HeLa cells.
Using anti-SNAP antibody, immunoblotting assay makes it possible to detect extracellular SNAP labels interferon (see Fig. 7 B).
3. the purifying of recombinant protein
With His labels and SNAP label proteins outside metal chelation resin and HLPC method purifying cells.
And TOS-N 3.1.RVF-N
RVF-N
Joint Plate-Forme 5Production de Prot é ines recombinants et d ' Anticorps
(Institute Pasteur), from Cd2+2 liters of S2/sSNAP-RVFV.N-His after stimulating 10 daysLabelIt is up in cell conditioned medium
97mg high-purity SNAP- labels RVF.N albumen.
TOS-N
Joint Plate-Forme 5Production de Prot é ines recombinants et d ' Anticorps
(Institute Pasteur), from Cd2+2 liters of S2/sSNAP-TOS.N-His after stimulating 10 daysLabelIt is up in cell conditioned medium
41mg high-purity SNAP- labels RVF.N albumen.
The summary of the level of production of table 1.：
3.2. solvable INF α 1
(Novagen kits) is cut by using enterokinase, the solvable albumen of INF α 1 is discharged from SNAP labels.
3.3. the antigen from different flavivirus
The SNAP- label proteins liquid storage (stock) that table 2. is secreted using Drosophila expression system
·EDIII：Domain antigenicity III (Dengue [DEN], Xi Niluo [WN], Japanese brain from flavivirus E protein
Scorching [JE], Usu figure [USU], tick propagate encephalitis [TBE], yellow hot [YF], Murray encephalitis [MVE], Wei Saiersi Blang [WSL],
Sieve Theo, hereby card)
·ectoM：The ectodomain of M albumen from I type dengue virus
N genes from RVF：The nucleoprotein N (main viral antigen) of Rift Valley fever virus
N genes from TOS：The nucleoprotein N (main viral antigen) of toscana virus
SE from WN：The soluble form of envelope E protein from west nile virus
SE2 from CHIK：The soluble form of Envelope 2 protein from chikungunya virus
·SNAP-IFNAI：The Alpha-IFN 1 merged with SNAP.
3.4. granzyme M production
In 7 days, 10mg SNAP-GrM albumen is reclaimed per L culture supernatants.
After purification step, the SNAP-GrM (referring to Fig. 6 C) of three kinds of forms is detected, the GrM enzymes progress of coupling is correspond to
The cutting of SNAP albumen.
Obviously, it means that by the inventive method produce after human protease be active (seeing below).
The control of 4.SNAP- label proteins
Immunoblotting assay detection using specific antibody (identification protein of interest and/or His labels are marked) is extracellular
The substantive production of SNAP- label proteins：
Immunoblotting assay uses the anti-His of sheep blood serumLabelDetect extracellular SNAP- labels RVF.N albumen (Fig. 2 B).It is anti-
RVF.N people and mouse immune serum specific recognition restructuring SNAP label RVF.N albumen.
Immunoblotting assay using specific mouse polyclonal serum shows, although sequence similarity level is high, restructuring
There is no cross reactivity between WN and USU EDIII.Therefore, the soluble SNAP- for coming from WNV, JE, USU of secretion
EDIII is suitable as recombinant virus antigens, for the member of specific diagnosis JE serum complexes, because in lesser degree,
USU and JE viruses are accredited as potential emerging arboviruse in Europe recently.
The activity of 5.SNAP- label recombinant proteins
Solvable restructuring SNAP-IFN α I secreted by the S2/SNAP-IFN α I cells of induction show powerful resist
CHIKV antiviral effect.
10 days after induction, Cd is collected2+S2/SNAP-IFN α I (#5 × 10^6 cells/ml) supernatant (5ml) of stimulation.Pass through
Observe that the accumulation of solvable SNAP-IFN α I proteins (is seen below in cell conditioned medium using the Western blotting of anti-His tag antibodies
Text).On the HeLa cells of the chikungunya virus infected with expressing luciferase (Luc) gene, assess SNAP-IFN α I's
Antiviral activity.Determine Luc activity within 6 hours after infection.(Infergen (Infergen) is used as interior part to IFN alphacon 1
Analysis, it is known that its powerful anti-CHIKV in HeLa cells antiviral effect.Cd2+The S2/SNAP-Tos.N of stimulation is (from support
The N protein of Si Kana viruses) supernatant is used as negative control.The SNAP-IFN α I or 0.1 μ g of the bright 1 μ l secretions of chart shown in Fig. 4 C
Infergen can suppress duplications of the CHIKV in host cells infected.SNAP-IFN α dosage dependent effect is as shown in FIG. 4 C.
Still observe that 20% Luc is active with the solvable SNAP-IFN α I of 0.1 μ l or 0.01 μ g Infergens.Under higher proof load,
Antiviral effect is not observed using SNAP-TOS-N.
Once being produced in S2 cell conditioned mediums, granzyme M is active
As it was previously stated, being detected in the S2 cell conditioned mediums that transfection has carrier pMT/BiP/SNAP-GrM-His labels
SNAP-GrM three kinds of forms (see Fig. 6 C).
The cutting for the SNAP albumen that the GrM enzymes that these three forms correspond to coupling are carried out.SNAP is really potential comprising three
GrM cleavage sites (see Fig. 6 B).Shown with the immunoassay of anti-His- or anti-SNAP antibody, these three forms are strictly
The fusion protein S NAP-GrM of secretion fragment.
Smaller form (35kDa) correspond to the deleted GrM of SNAP major parts in purge process.
These results are clearly illustrated, although coupling has SNAP albumen, is as the GrM protease produced by present system
It is active.
This is really interesting, because it is known that active human protease is difficult by recombinant production.
Once produced in the cell conditioned mediums of HEK 293, hSULF-2ΔTMDIt is active
hSULF-2ΔTMDExpressing and purifying has recombinant plasmid pcDNA3/De SNAPuniv-hSULF-2 in transfectionΔTMD's
HEK-293 cells (see Figure 13 A).
According to the following hSULF-2 evaluated obtained from supernatantΔTMDThe enzymatic activity of polypeptide：
The cell transient transfections of HEK 293 have pcDNA3/DeSNAP-hSULF2 Δs TMD.Two days later, the equal portions of cell conditioned medium
It is incubated with the pseudo- substrate 4-methyl umbelliferone (Umbelliferone) (4-MUS) of 20mM non-fluorescence in 50mM Tris pH7.5,
20mM magnesium chlorides (1:1, V/V) it is incubated 2-4 hours at 37 DEG C in 96- orifice plates with enzyme (in conditioned medium).By adding
Plus (1:1v/v) 1.5M NaHCO3/Na2CO3pH 10.5 terminate enzyme reaction, and (are excited by fluorescence photometer：360nm) monitor 4-
The generation of methyl umbelliferone fluorescence-causing substance.With the value of SULF activity in optical density (OD) the measurement cell conditioned medium at 460nm.
It is interesting that as shown in Figure 13 B, the albumen (coupling has SNAP) of secretion is active.
The stability of 6.SNAP label recombinant proteins
It has been surprisingly observed that, include the fusion protein of SNAP peptides, it is more stable in vitro during than no SNAP peptides.
Highly purified CHIK.sE2-SNAP, SNAP-WN.EDIII and SNAP-IFNAI albumen, in sterile PBS
0.1mg/ml(Vol：0.1ml) during unsaturation concentration, it is incubated 4 days at -80 DEG C, 4 DEG C, 25 DEG C or 37 DEG C, or mutually synthermal
It is lower to be incubated two months.
Protein isolate sample (1 μ g) on SDS-PAGE 4-12%, and use PageBlue protein staining solution
(Fermentas) visualized with Coomassie brilliant G-250 dyestuff.
Figure 10 A to 10D are disclosed by comparing the result that the vitro stability of three kinds of different fusion proteins is obtained.
After being incubated four days importantly, at 4 DEG C after 2 months, and at room temperature at (25 DEG C) or 37 DEG C, all fusions
Albumen is seemingly complete.
Particularly, under body temperature (37 DEG C), IFN is unaffected after 4 days, and still observed after 2 months at 37 DEG C
Arrive, therefore have SNAP by its coupling, internal stability still may be improved highly.
The vitro detection for the SNAP-RVF.N that 7.S2 cells are produced
The SNAP-RVF.N fusion proteins produced according to above-mentioned code are used as diagnostic tool, the Sheep Blood for detecting infection
Anti- RVF.N antibody in clear.
These fusion proteins are detected, and compare (many things of RVF from BDSL and from IdVet with commercial detection kit
The RVF IgG detection kits planted).
Tested on 46 parts of sheep serums, sheep immunity inoculation there are RVF vaccines.SNAP-RVF.N fusion proteins are direct
Bottom hole is coated on, or through biotinylation and is added in the coated hole of Streptavidin.
By indirect ELISA method, there is high-purity recombinant antigen SNAP-RVF.N of 0.2 μ g in PBS (dense using direct coated
Degree：2 μ g albumen/ml) microtitration 96- holes patch, at 4 DEG C overnight detect anti-RVF antibody.After saturation, the serum of dilution
It is incubated with SNAP-RVF.N.Coupling has goat-anti-IgG peroxidase as secondary antibody.Carried out with Peroxidase Substrate System
ELISA, measures optical density (OD) at 450nm.If OD is twice of the OD from non-immune serum, then it is assumed that sample serum
It is positive.
It is interesting that as a result showing when in direct coated to hole, compared to business albumen, SNAP-RVF.N fusion proteins
Provide identical sensitivity and specificity (not shown).When albumen is biotinylated, reappearance as a result is relatively low.
On the cow's serum obtained from the innate immunity, achieve identical result (data are not shown).
These results show that the fusion protein of the present invention can be used as being used to identify virus infection or bacterium sense in biological specimen
The diagnostic tool of dye.
8. the multiple immunoassay based on pearl
In the context of the present invention, developing the multiple immunoassay based on pearl is used for quick and synchronous detection biological fluid
The antibody of moderate resistance arboviruse.
The system is based on xMAP technologies (Luminex companies), and using the mixture of microspheres of antigen is coated with as specific
The capturing agent of human immunoglobulin(HIg).Different groups of microballoon (Magplex, Luminex public affairs are coupled with the MGMT fusion proteins of purifying
Department), i.e., the viral recombinant protein of SNAP- labels described in 3.3 sections：sSNAP-DV1.EDIII、sSNAP-DV2.EDIII、
sSNAP-DV3.EDIII、sSNAP-DV4.EDIII、sSNAP-WN.EDIII、sSNAP-JE.EDIII、sSNAP-
USU.EDIII、sSNAP-TBE.EDIII、sSNAP-YF.EDIII、sSNAP-MVE.EDIII、sSNAP-Rocio.EDIII、
SSNAP-WSL.EDIII, sSNAP-ZIKA.EDIII, SNAP-DV1ectoM, sSNAP-N.RVF, sSNAP-N.TOS and
CHIK.sE2-SNAP.With the substrate of mgmt protein as joint (BG-PEG-NH2, New England Biolabs), it will recombinate
Antigen is covalently coupled to carboxyl microsphere surface, so as to compared with standard amine coupling procedure, strengthen antibody capture efficiency.
Technical identification using anti-SNAP- tag antibodies and specific mouse monoclonal antibody confirms coupling efficiency, and
Indicate long-term Antigen Stability (being up to 6 months).The application is not limited to viral antigen, because any peptide or polypeptide can be used for
The coating and subsequent antibody capture of pearl.
Bibliography
Ausubel F.M. et al. (editor), Current Protocols in Molecular Biology, John
Wiley&Sons,Inc.(1994).
Bond B.J. et al., Mol.Cell.Biol.6:2080(1986)
Brehin et al., Virology 371:185,2008
Brinster et al., Nature, 296:39-42,1982
Cheever et al., Clinical Cancer Research, 2009,15:5323-5337
Dai et al., the Journal of Biological Chemistry, 2005, vol.280.n ° 48,
pp.40066-40073
Daniels D.S. et al., EMBO are J.19:1719-1730,2000
Felgner et al., Proc.Natl.Acad.Sci.U.S.A., 84:7413-7417,1987
Harlowe, et al., 1988, Antibodies:A Laboratory Manual,Cold Spring Harbor
Laboratory Press
Hellwig S et al., Nature Biotechnology, n ° 11, vol.22,2004
Hu D. et al., 2010The Journal of Biological Chemistry, vol.285, n ° 24,
pp18326-18335
Juillerat A. et al., Chemistry＆Biology, vol.10,313-317,2003
Lastowski-Perry et al., J.Biol.Chem.260:1527(1985)
Lim A. et al., EMBO are J.15:4050-4060,1996
Ludolfs et al., 2007Eur J Clin Microbiol Infect Dis.2007Jul；26(7):467-73
Ma JKC et al., Nature Genetics, Review 2004,794-800
Mackey et al., Proc.Natl.Acad.Sci.U.S.A., 85:8027-8031,1988
Miller and Rosman,BioTechniques,7:980-990,1992
Mokkim et al. protein expression purif.63:140,2009.
Pegg A.E. et al., Mutat.Res.2000；462,82-100
Perbal B.E.,A Practical Guide to Molecular Cloning(1984)；
Sambrook,Fitsch&Maniatis,Molecular Cloning:A Laboratory Manual,Second
Edition(1989)Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.
(referred to herein as " Sambrook et al., 1989 ")；
Shimoda Y.and Watanabe K.,Cell adhesion and migration,2009,3:1,64-70
Van Domselaar R. et al., The Journal of Immunology, 2010
Wibley et al., Nucleic acid research 2000
Williams et al., Proc.Natl.Acad.Sci.U.S.A., 88:2726-2730,1991
Wu and Wu,J.Biol.Chem.,263:14621-14624,1988；
Wu et al., J.Biol.Chem., 267:963-967,1992；
DNA Cloning:(D.N.Glover is edited A Practical Approach, Volumes I and II
1985)；Oligonucleotide Synthesis (M.J.Gait edits 1984)；
Nucleic Acid Hybridization (B.D.Hames＆S.J.Higgins edits 1984)；
(R.I.Freshney, editor is 1986) by Animal Cell Culture；
Immobilized Cells and Enzymes(IRL Press,1986)。
Sequence table
<110>Institute Pasteur
<120>The method based on MGMT for obtaining high yield expression of recombinant proteins
<130> 359961D29054
<150> EP 10306389.7
<151> 2010-12-09
<150> US 61/505,694
<151> 2011-07-08
<160> 97
<170> PatentIn version 3.5
<210> 1
<211> 579
<212> DNA
<213>Artificial sequence
<220>
<223>SNAP DNA (hMGT mutant)
<400> 1
agatctgaca aagactgcga aatgaagcgc accaccctgg atagccctct gggcaagctg 60
gaactgtctg ggtgcgaaca gggcctgcac gagatcaagc tgctgggcaa aggaacatct 120
gccgccgacg ccgtggaagt gcctgcccca gccgccgtgc tgggcggacc agagccactg 180
atgcaggcca ccgcctggct caacgcctac tttcaccagc ctgaggccat cgaggagttc 240
cctgtgccag ccctgcacca cccagtgttc cagcaggaga gctttacccg ccaggtgctg 300
tggaaactgc tgaaagtggt gaagttcgga gaggtcatca gctaccagca gctggccgcc 360
ctggccggca atcccgccgc caccgccgcc gtgaaaaccg ccctgagcgg aaatcccgtg 420
cccattctga tcccctgcca ccgggtggtg tctagctctg gcgccgtggg gggctacgag 480
ggcgggctcg ccgtgaaaga gtggctgctg gcccacgagg gccacagact gggcaagcct 540
gggctgggtc ctgcaggtat aggcgcgcca gggtcccta 579
<210> 2
<211> 193
<212> PRT
<213>Artificial sequence
<220>
<223>SNAP amino acid sequence
<400> 2
Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp Ser Pro
1 5 10 15
Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His Glu Ile
20 25 30
Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu Val Pro
35 40 45
Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln Ala Thr
50 55 60
Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu Glu Phe
65 70 75 80
Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser Phe Thr
85 90 95
Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly Glu Val
100 105 110
Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala Ala Thr
115 120 125
Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile Leu Ile
130 135 140
Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly Tyr Glu
145 150 155 160
Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly His Arg
165 170 175
Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro Gly Ser
180 185 190
Leu
<210> 3
<211> 1265
<212> DNA
<213>People
<400> 3
ctcggccccg cccccgcgcc ccggatatgc tgggacagcc cgcgccccta gaacgctttg 60
cgtcccgacg cccgcaggtc ctcgcggtgc gcaccgtttg cgacttggta cttggaaaaa 120
tggacaagga ttgtgaaatg aaacgcacca cactggacag ccctttgggg aagctggagc 180
tgtctggttg tgagcagggt ctgcacgaaa taaagctcct gggcaagggg acgtctgcag 240
ctgatgccgt ggaggtccca gcccccgctg cggttctcgg aggtccggag cccctgatgc 300
agtgcacagc ctggctgaat gcctatttcc accagcccga ggctatcgaa gagttccccg 360
tgccggctct tcaccatccc gttttccagc aagagtcgtt caccagacag gtgttatgga 420
agctgctgaa ggttgtgaaa ttcggagaag tgatttctta ccagcaatta gcagccctgg 480
caggcaaccc caaagccgcg cgagcagtgg gaggagcaat gagaggcaat cctgtcccca 540
tcctcatccc gtgccacaga gtggtctgca gcagcggagc cgtgggcaac tactccggag 600
gactggccgt gaaggaatgg cttctggccc atgaaggcca ccggttgggg aagccaggct 660
tgggagggag ctcaggtctg gcaggggcct ggctcaaggg agcgggagct acctcgggct 720
ccccgcctgc tggccgaaac tgagtatgtg cagtaggatg gatgtttgag cgacacacac 780
gtgtaacact gcatcggatg cggggcgtgg aggcaccgct gtattaaagg aagtggcagt 840
gtcctgggaa caagcgtgtc tgccctttct gtttccatat tttacagcag gatgagttca 900
gacgcccgcg gtcctgcaca catttgtttc cttctctaac gctgcccttg ctctattttt 960
catgtccatt aaaacaggcc aagtgagtgt ggaaggcctg gctcatgttg ggccacagcc 1020
caggatgggg cagtctggca ccctcaggcc acagacggct gccatagccg ctgtccaggg 1080
ccagctaagg cccatcccag gccgtccaca ctagaaagct ggccctgccc catccccacc 1140
atgcctccct tcctggctgt gtccatggct gtgatggcat tctccactca gcagttccta 1200
gcatcccaca cccaggtctc actgaaagaa aggggaacag gccatggcag tcagtgctta 1260
cagag 1265
<210> 4
<211> 238
<212> PRT
<213>People
<400> 4
Met Leu Gly Gln Pro Ala Pro Leu Glu Arg Phe Ala Ser Arg Arg Pro
1 5 10 15
Gln Val Leu Ala Val Arg Thr Val Cys Asp Leu Val Leu Gly Lys Met
20 25 30
Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp Ser Pro Leu Gly
35 40 45
Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His Glu Ile Lys Leu
50 55 60
Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu Val Pro Ala Pro
65 70 75 80
Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln Cys Thr Ala Trp
85 90 95
Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu Glu Phe Pro Val
100 105 110
Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser Phe Thr Arg Gln
115 120 125
Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly Glu Val Ile Ser
130 135 140
Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Lys Ala Ala Arg Ala
145 150 155 160
Val Gly Gly Ala Met Arg Gly Asn Pro Val Pro Ile Leu Ile Pro Cys
165 170 175
His Arg Val Val Cys Ser Ser Gly Ala Val Gly Asn Tyr Ser Gly Gly
180 185 190
Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly His Arg Leu Gly
195 200 205
Lys Pro Gly Leu Gly Gly Ser Ser Gly Leu Ala Gly Ala Trp Leu Lys
210 215 220
Gly Ala Gly Ala Thr Ser Gly Ser Pro Pro Ala Gly Arg Asn
225 230 235
<210> 5
<211> 713
<212> DNA
<213>Artificial sequence
<220>
<223> DNA sequence of BiP + SNAP+ enterokinase site + EcoRV/XmaI+Histag
<400> 5
atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct cgggagatct 60
gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc ctctgggcaa gctggaactg 120
tctgggtgcg aacagggcct gcacgagatc aagctgctgg gcaaaggaac atctgccgcc 180
gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg gaccagagcc actgatgcag 240
gccaccgcct ggctcaacgc ctactttcac cagcctgagg ccatcgagga gttccctgtg 300
ccagccctgc accacccagt gttccagcag gagagcttta cccgccaggt gctgtggaaa 360
ctgctgaaag tggtgaagtt cggagaggtc atcagctacc agcagctggc cgccctggcc 420
ggcaatcccg ccgccaccgc cgccgtgaaa accgccctga gcggaaatcc cgtgcccatt 480
ctgatcccct gccaccgggt ggtgtctagc tctggcgccg tggggggcta cgagggcggg 540
ctcgccgtga aagagtggct gctggcccac gagggccaca gactgggcaa gcctgggctg 600
ggtcctgcag gtataggcgc gccagggtcc ctaggtggcg gatctgatga cgatgataaa 660
gatatcaaaa acccgggcgg tggaagtcat catcatcatc atcattgacc ggt 713
<210> 6
<211> 614
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of SNAP+His labels
<400> 6
agatctgaca aagactgcga aatgaagcgc accaccctgg atagccctct gggcaagctg 60
gaactgtctg ggtgcgaaca gggcctgcac gagatcaagc tgctgggcaa aggaacatct 120
gccgccgacg ccgtggaagt gcctgcccca gccgccgtgc tgggcggacc agagccactg 180
atgcaggcca ccgcctggct caacgcctac tttcaccagc ctgaggccat cgaggagttc 240
cctgtgccag ccctgcacca cccagtgttc cagcaggaga gctttacccg ccaggtgctg 300
tggaaactgc tgaaagtggt gaagttcgga gaggtcatca gctaccagca gctggccgcc 360
ctggccggca atcccgccgc caccgccgcc gtgaaaaccg ccctgagcgg aaatcccgtg 420
cccattctga tcccctgcca ccgggtggtg tctagctctg gcgccgtggg gggctacgag 480
ggcgggctcg ccgtgaaaga gtggctgctg gcccacgagg gccacagact gggcaagcct 540
gggctgggtc ctgcaggtat aggcgcgcca gggtccctgg agcatcatca tcatcatcat 600
tgatgagcgg ccgc 614
<210> 7
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 5'-SNAP
<400> 7
aaaaaagatc tgacaaagac tgcgaaatg 29
<210> 8
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 3'-MCS (SNAP)
<400> 8
gaggagaggg ttagggatag gcttacc 27
<210> 9
<211> 4181
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna (DeSNAP1) of carrier of the present invention
<400> 9
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccag tgaattttaa cgttgcagga 420
caggatgtgg tgcccgatgt gactagctct ttgctgcagg ccgtcctatc ctctggttcc 480
gataagagac ccagaactcc ggccccccac cgcccaccgc cacccccata catatgtggt 540
acgcaagtaa gagtgcctgc gcatgcccca tgtgccccac caagagtttt gcatcccata 600
caagtcccca aagtggagaa ccgaaccaat tcttcgcggg cagaacaaaa gcttctgcac 660
acgtctccac tcgaatttgg agccggccgg cgtgtgcaaa agaggtgaat cgaacgaaag 720
acccgtgtgt aaagccgcgt ttccaaaatg tataaaaccg agagcatctg gccaatgtgc 780
atcagttgtg gtcagcagca aaatcaagtg aatcatctca gtgcaactaa aggggggatc 840
cgatctcaat atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct 900
cgggagatct gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc ctctgggcaa 960
gctggaactg tctgggtgcg aacagggcct gcacgagatc aagctgctgg gcaaaggaac 1020
atctgccgcc gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg gaccagagcc 1080
actgatgcag gccaccgcct ggctcaacgc ctactttcac cagcctgagg ccatcgagga 1140
gttccctgtg ccagccctgc accacccagt gttccagcag gagagcttta cccgccaggt 1200
gctgtggaaa ctgctgaaag tggtgaagtt cggagaggtc atcagctacc agcagctggc 1260
cgccctggcc ggcaatcccg ccgccaccgc cgccgtgaaa accgccctga gcggaaatcc 1320
cgtgcccatt ctgatcccct gccaccgggt ggtgtctagc tctggcgccg tggggggcta 1380
cgagggcggg ctcgccgtga aagagtggct gctggcccac gagggccaca gactgggcaa 1440
gcctgggctg ggtcctgcag gtataggcgc gccagggtcc ctggagcatc atcatcatca 1500
tcattgatga gcggccgctc gagtctagag ggcccttcga aggtaagcct atccctaacc 1560
ctctcctcgg tctcgattct acgcgtaccg gtcatcatca ccatcaccat tgagtttaaa 1620
cccgctgatc agcctcgact gtgccttcta aggcctgagc tcgctgatca gcctcgatcg 1680
aggatccaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt 1740
gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa 1800
gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg 1860
aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg 1920
atcagtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt 1980
gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 2040
cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 2100
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 2160
gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 2220
ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 2280
caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 2340
aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 2400
atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 2460
cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 2520
ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 2580
gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 2640
accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 2700
cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 2760
cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 2820
gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 2880
aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 2940
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 3000
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 3060
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 3120
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 3180
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 3240
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 3300
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 3360
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 3420
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 3480
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 3540
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 3600
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 3660
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 3720
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 3780
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 3840
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 3900
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 3960
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 4020
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 4080
ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga 4140
cattaaccta taaaaatagg cgtatcacga ggccctttcg t 4181
<210> 10
<211> 1720
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of DeSNAP2 carriers of the present invention
<400> 10
gaattcgttg caggacagga tgtggtgccc gatgtgacta gctctttgct gcaggccgtc 60
ctatcctctg gttccgataa gagacccaga actccggccc cccaccgccc accgccaccc 120
ccatacatat gtggtacgca agtaagagtg cctgcgcatg ccccatgtgc cccaccaaga 180
gttttgcatc ccatacaagt ccccaaagtg gagaaccgaa ccaattcttc gcgggcagaa 240
caaaagcttc tgcacacgtc tccactcgaa tttggagccg gccggcgtgt gcaaaagagg 300
tgaatcgaac gaaagacccg tgtgtaaagc cgcgtttcca aaatctataa aaccgagagc 360
atctggacca tgtgcatcag ttgtggtcag cagcaaaatc aagtgaatca tctcagtgca 420
actaaagggg ggatccgatc tcaatgcgag ctgtttctta gcacgaagat ctcgatgtct 480
aagaaaccag gagggccggg caagagccgg gctgtcaata ccatggttgt gtttgtcgtg 540
ctattgcttt tggtggcccc agcttacagc cttgatattg aatttacaga caaagactgc 600
gaaatgaaaa gaactacatt ggattcacca cttgggaagt tggaactgag tggatgcgag 660
caaggattgc atgaaattaa gcttctggga aaaggaactt ctgcagctga tgcagttgaa 720
gttccagcac cagcagctgt tcttggaggt cctgagcccc tcatgcaagc cacagcctgg 780
cttaacgcat atttccacca gcctgaggcc attgaggaat ttccagtccc cgcccttcac 840
catcctgtgt ttcagcagga aagcttcacc cgccaggtcc tgtggaaatt gctgaaggtg 900
gtcaagtttg gtgaagtgat ttcatatcag caacttgctg cattggccgg taaccccgca 960
gctacagctg ccgtgaaaac tgctctcagc ggaaatcctg tgcccatcct gatcccttgt 1020
cacagagtcg tttcatcttc cggagctgta ggtggctatg aaggaggact ggcagttaag 1080
gagtggctgc tggctcatga aggtcataga cttggaaaac ctggtttggg aggtggcgga 1140
tctgatgacg atgataaaga tatcatatac ccgggcggtg gaagtcatca tcaccatcac 1200
cactgataaa tatttaatca attgtaaata gacaatataa gtatgcataa aagtgtagtt 1260
ttatagtagt atttagtggt gttagtgtaa atagttaaga aaattttgag gagaaagtca 1320
ggccgggaag ttcccgccac cggaagttga gtagacggtg ctgcctgcga ctcaacccca 1380
ggaggactgg gtgaacaaag ccgcgaagtg atccatgtaa gccctcagaa ccgtctcgga 1440
aggaggaccc cacatgttgt aacttcaaag cccaatgtca gaccacgcta cggcgtgcta 1500
ctctgcggag agtgcagtct gcgatagtgt aacaaaggca aatcaacgcc ccacgcggcc 1560
ctagccccgg taatggtgtt aaccagggcg aaaggactag aggttagagg agaccccgcg 1620
gtttaaagtg cacggcccag cctggctgaa gctgtaggtc aggggtggag accccgtgcc 1680
acaaaacacc acaacaaaac agcataaata aacagggccc 1720
<210> 11
<211> 54
<212> DNA
<213>Artificial sequence
<220>
<223>Encode the DNA sequence dna of ssBiP sequences
<400> 11
atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct cggg 54
<210> 12
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Encode the DNA sequence dna of enterokinase cleavage site point
<400> 12
gatgacgatg ataaagat 18
<210> 13
<211> 12
<212> DNA
<213>Artificial sequence
<220>
<223>DNA intervening sequences
<400> 13
ggtggcggat ct 12
<210> 14
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Encode the DNA sequence dna of His labels
<400> 14
catcatcatc atcatcat 18
<210> 15
<211> 17
<212> PRT
<213>West nile virus strain IS-98-ST1
<400> 15
Met Val Val Phe Val Val Leu Leu Leu Leu Val Ala Pro Ala Tyr Ser
1 5 10 15
Leu
<210> 16
<211> 1361
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of SNAP/RVF.N/His labels
<400> 16
agatctgaca aagactgcga aatgaagcgc accaccctgg atagccctct gggcaagctg 60
gaactgtctg ggtgcgaaca gggcctgcac gagatcaagc tgctgggcaa aggaacatct 120
gccgccgacg ccgtggaagt gcctgcccca gccgccgtgc tgggcggacc agagccactg 180
atgcaggcca ccgcctggct caacgcctac tttcaccagc ctgaggccat cgaggagttc 240
cctgtgccag ccctgcacca cccagtgttc cagcaggaga gctttacccg ccaggtgctg 300
tggaaactgc tgaaagtggt gaagttcgga gaggtcatca gctaccagca gctggccgcc 360
ctggccggca atcccgccgc caccgccgcc gtgaaaaccg ccctgagcgg aaatcccgtg 420
cccattctga tcccctgcca ccgggtggtg tctagctctg gcgccgtggg gggctacgag 480
ggcgggctcg ccgtgaaaga gtggctgctg gcccacgagg gccacagact gggcaagcct 540
gggctgggtc ctgcaggtat aggcgcgcca gggtccctag gtggcggatc tgacaactat 600
caagagcttc gagtccagtt tgctgctcaa gcagtggacc gcaatgagat tgaacagtgg 660
gtccgagagt ttgcttatca agggtttgat gcccgtagag ttatcgaact cttaaagcag 720
tatggtgggg ctgactggga gaaggatgcc aagaaaatga ttgttctggc tctaactcgt 780
ggcaacaagc ccaggaggat gatgatgaaa atgtcgaaag aaggcaaagc aactgtggag 840
gctctcatca acaagtataa gctaaaggaa gggaatcctt cccgggatga gttgactcta 900
tcacgagttg ctgccgcctt ggctggctgg acatgccagg ctttggtcgt cttgagtgag 960
tggcttcctg tcactgggac taccatggac ggcctatccc ctgcataccc gaggcatatg 1020
atgcacccca gctttgctgg catggtggat ccttctctac caggagacta tctaagggca 1080
atattagatg ctcactctct gtatctgctg cagttctccc gggtcatcaa cccaaacctc 1140
cgaggtagaa caaaagagga ggttgctgca acgttcacgc agccaatgaa tgcagcagtg 1200
aatagcaact ttataagcca tgagaagagg agagaattct tgaaagcctt tggacttgtg 1260
gattccaatg ggaagccgtc agctgctgtc atggcagccg ctcaggctta caagacagca 1320
gccggcggtg gaagtcatca tcatcatcat cattgaccgg t 1361
<210> 17
<211> 66
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 5'N (is used to clone RVF-N)
<400> 17
aaaaaggcgc gccagggggt ggcggatctg acaactatca agagcttcga gtccagtttg 60
ctgctc 66
<210> 18
<211> 69
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 3'N (is used to clone RVF-N)
<400> 18
aaaaaaccgg tcaatgatga tgatgatgat gacttccacc gccggctgct gtcttgtaag 60
cctgagcgg 69
<210> 19
<211> 4854
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of pMT/BiP/SNAP-RVF.N/His labels
<400> 19
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccag tgaattttaa cgttgcagga 420
caggatgtgg tgcccgatgt gactagctct ttgctgcagg ccgtcctatc ctctggttcc 480
gataagagac ccagaactcc ggccccccac cgcccaccgc cacccccata catatgtggt 540
acgcaagtaa gagtgcctgc gcatgcccca tgtgccccac caagagtttt gcatcccata 600
caagtcccca aagtggagaa ccgaaccaat tcttcgcggg cagaacaaaa gcttctgcac 660
acgtctccac tcgaatttgg agccggccgg cgtgtgcaaa agaggtgaat cgaacgaaag 720
acccgtgtgt aaagccgcgt ttccaaaatg tataaaaccg agagcatctg gccaatgtgc 780
atcagttgtg gtcagcagca aaatcaagtg aatcatctca gtgcaactaa aggggggatc 840
cgatctcaat atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct 900
cgggagatct gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc ctctgggcaa 960
gctggaactg tctgggtgcg aacagggcct gcacgagatc aagctgctgg gcaaaggaac 1020
atctgccgcc gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg gaccagagcc 1080
actgatgcag gccaccgcct ggctcaacgc ctactttcac cagcctgagg ccatcgagga 1140
gttccctgtg ccagccctgc accacccagt gttccagcag gagagcttta cccgccaggt 1200
gctgtggaaa ctgctgaaag tggtgaagtt cggagaggtc atcagctacc agcagctggc 1260
cgccctggcc ggcaatcccg ccgccaccgc cgccgtgaaa accgccctga gcggaaatcc 1320
cgtgcccatt ctgatcccct gccaccgggt ggtgtctagc tctggcgccg tggggggcta 1380
cgagggcggg ctcgccgtga aagagtggct gctggcccac gagggccaca gactgggcaa 1440
gcctgggctg ggtcctgcag gtataggcgc gccagggtcc ctaggtggcg gatctgacaa 1500
ctatcaagag cttcgagtcc agtttgctgc tcaagcagtg gaccgcaatg agattgaaca 1560
gtgggtccga gagtttgctt atcaagggtt tgatgcccgt agagttatcg aactcttaaa 1620
gcagtatggt ggggctgact gggagaagga tgccaagaaa atgattgttc tggctctaac 1680
tcgtggcaac aagcccagga ggatgatgat gaaaatgtcg aaagaaggca aagcaactgt 1740
ggaggctctc atcaacaagt ataagctaaa ggaagggaat ccttcccggg atgagttgac 1800
tctatcacga gttgctgccg ccttggctgg ctggacatgc caggctttgg tcgtcttgag 1860
tgagtggctt cctgtcactg ggactaccat ggacggccta tcccctgcat acccgaggca 1920
tatgatgcac cccagctttg ctggcatggt ggatccttct ctaccaggag actatctaag 1980
ggcaatatta gatgctcact ctctgtatct gctgcagttc tcccgggtca tcaacccaaa 2040
cctccgaggt agaacaaaag aggaggttgc tgcaacgttc acgcagccaa tgaatgcagc 2100
agtgaatagc aactttataa gccatgagaa gaggagagaa ttcttgaaag cctttggact 2160
tgtggattcc aatgggaagc cgtcagctgc tgtcatggca gccgctcagg cttacaagac 2220
agcagccggc ggtggaagtc atcatcatca tcatcattga ccggtcatca tcaccatcac 2280
cattgagttt aaacccgctg atcagcctcg actgtgcctt ctaaggcctg agctcgctga 2340
tcagcctcga tcgaggatcc agacatgata agatacattg atgagtttgg acaaaccaca 2400
actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt 2460
gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt 2520
caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt 2580
atggctgatt atgatcagtc gacctgcagg catgcaagct tggcgtaatc atggtcatag 2640
ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 2700
ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 2760
tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 2820
cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 2880
ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 2940
ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 3000
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 3060
gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 3120
taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 3180
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 3240
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 3300
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 3360
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 3420
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 3480
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 3540
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 3600
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 3660
cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 3720
acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 3780
acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 3840
tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 3900
ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 3960
ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 4020
tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 4080
aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 4140
ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 4200
ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 4260
gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 4320
gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 4380
cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 4440
actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 4500
ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 4560
tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 4620
ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 4680
agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4740
aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 4800
attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgt 4854
<210> 20
<211> 1957
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of WNsE/SNAP/His labels
<400> 20
agatctttca actgccttgg aatgagcaac agagacttct tggaaggagt gtctggagca 60
acatgggtgg atttggttct cgaaggcgac agctgcgtga ctatcatgtc taaggacaag 120
cctaccatcg atgtgaagat gatgaatatg gaggcggcca acctggcaga ggtccgcagt 180
tattgctatt tggctaccgt cagcgatctc tccaccaaag ctgcgtgccc gaccatggga 240
gaagctcaca atgacaaacg tgctgaccca gcttttgtgt gcagacaagg agtagtggac 300
aggggctggg gcaacggctg cggactattt ggcaaaggaa gcattgacac atgcgccaaa 360
tttgcctgct ctaccaaggc aataggaaga accatcttga aagagaatat caagtacgaa 420
gtggccattt ttgtccatgg accaactact gtggagtcgc acggaaacta ctccacacag 480
gttggagcca ctcaggcagg gagattcagc atcactcctg cggcgccttc atacacacta 540
aagcttggag aatatggaga ggtgacagtg gactgtgaac cacggtcagg gattgacacc 600
aatgcatact acgtgatgac tgttggaaca aagacgttct tggtccatcg tgagtggttc 660
atggacctca acctcccttg gagcagtgct ggaagtactg tgtggaggaa cagagagacg 720
ttaatggagt ttgaggaacc acacgccacg aagcagtctg tgatagcatt gggctcacaa 780
gagggagctc tgcatcaagc tttggctgga gccattcctg tggaattttc aagcaacact 840
gtcaagttga cgtcgggtca tttgaagtgt agagtgaaga tggaaaaatt gcagttgaag 900
ggaacaacct atggcgtctg ttcaaaggct ttcaagtttc ttgggactcc cgcagacaca 960
ggtcacggca ctgtggtgtt ggaattgcag tacactggca cggatggacc ttgcaaagtt 1020
cctatctcgt cagtggcttc attgaacgac ctaacgccag tgggcagatt ggtcactgtc 1080
aacccttttg tttcagtggc cacggccaac gctaaggtcc tgattgaatt ggaaccaccc 1140
tttggagact catacatagt ggtgggcaga ggagaacaac agattaatca ccattggcac 1200
aagtctggaa gcagcattgg caaagccttt acaaccaccc tcaaaggagc gcagagacta 1260
gccgctctag gagacacagc ttgggacttt ggatcagttg gaggggtgtt cacctcagtt 1320
gggaaggctg tgcggccgct gggcggaggt agcaaagact gcgaaatgaa gcgcaccacc 1380
ctggatagcc ctctgggcaa gctggaactg tctgggtgcg aacagggcct gcacgagatc 1440
aagctgctgg gcaaaggaac atctgccgcc gacgccgtgg aagtgcctgc cccagccgcc 1500
gtgctgggcg gaccagagcc actgatgcag gccaccgcct ggctcaacgc ctactttcac 1560
cagcctgagg ccatcgagga gttccctgtg ccagccctgc accacccagt gttccagcag 1620
gagagcttta cccgccaggt gctgtggaaa ctgctgaaag tggtgaagtt cggagaggtc 1680
atcagctacc agcagctggc cgccctggcc ggcaatcccg ccgccaccgc cgccgtgaaa 1740
accgccctga gcggaaatcc cgtgcccatt ctgatcccct gccaccgggt ggtgtctagc 1800
tctggcgccg tggggggcta cgagggcggg ctcgccgtga aagagtggct gctggcccac 1860
gagggccaca gactgggcaa gcctgggctg ggtcctgcag gtataggcgc gccagggtcc 1920
ctggagcatc atcatcatca tcattgatga cgggccc 1957
<210> 21
<211> 960
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of BiP/SNAP-IFN/His labels
<400> 21
atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct cgggagatct 60
gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc ctctgggcaa gctggaactg 120
tctgggtgcg aacagggcct gcacgagatc aagctgctgg gcaaaggaac atctgccgcc 180
gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg gaccagagcc actgatgcag 240
gccaccgcct ggctcaacgc ctactttcac cagcctgagg ccatcgagga gttccctgtg 300
ccagccctgc accacccagt gttccagcag gagagcttta cccgccaggt gctgtggaaa 360
ctgctgaaag tggtgaagtt cggagaggtc atcagctacc agcagctggc cgccctggcc 420
ggcaatcccg ccgccaccgc cgccgtgaaa accgccctga gcggaaatcc cgtgcccatt 480
ctgatcccct gccaccgggt ggtgtctagc tctggcgccg tggggggcta cgagggcggg 540
ctcgccgtga aagagtggct gctggcccac gagggccaca gactgggcaa gcctgggctg 600
ggtcctgcag gtataggcgc gccagggtcc ctaggtggcg gatctgatga cgatgataaa 660
gatatctgtg atctccctga gacccacagc ctggataaca ggaggacctt gatgctcctg 720
gcacaaatga gcagaatctc tccttcctcc tgtctgatgg acagacatga ctttggattt 780
ccccaggagg agtttgatgg caaccagttc cagaaggctc cagccatctc tgtcctccat 840
gagctgatcc agcagatctt caacctcttt accacaaaag attcatctgc tgcttgggat 900
gaggacctcc tagacaaatt ctgcaccgaa ctctaccagc agctgaatga cttggaagcc 960
<210> 22
<211> 957
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of SNAP-EDIIIWN/His labels
<400> 22
agatctgaca aagactgcga aatgaagcgc accaccctgg atagccctct gggcaagctg 60
gaactgtctg ggtgcgaaca gggcctgcac gagatcaagc tgctgggcaa aggaacatct 120
gccgccgacg ccgtggaagt gcctgcccca gccgccgtgc tgggcggacc agagccactg 180
atgcaggcca ccgcctggct caacgcctac tttcaccagc ctgaggccat cgaggagttc 240
cctgtgccag ccctgcacca cccagtgttc cagcaggaga gctttacccg ccaggtgctg 300
tggaaactgc tgaaagtggt gaagttcgga gaggtcatca gctaccagca gctggccgcc 360
ctggccggca atcccgccgc caccgccgcc gtgaaaaccg ccctgagcgg aaatcccgtg 420
cccattctga tcccctgcca ccgggtggtg tctagctctg gcgccgtggg gggctacgag 480
ggcgggctcg ccgtgaaaga gtggctgctg gcccacgagg gccacagact gggcaagcct 540
gggctgggtc ctgcaggtat aggcgcgcca ggaggtggcg ggtctcagtt gaagggaaca 600
acctatggcg tctgttcaaa ggctttcaag tttcttggga ctcccgcaga cacaggtcac 660
ggcactgtgg tgttggaatt gcagtacact ggcacggatg gaccttgcaa agttcctatc 720
tcgtcagtgg cttcattgaa cgacctaacg ccagtgggca gattggtcac tgtcaaccct 780
tttgtttcag tggccacggc caacgctaag gtcctgattg aattggaacc accctttgga 840
gactcataca tagtggtggg cagaggagaa caacagatca atcaccattg gcacaagtct 900
ggaagcagca ttggcaaagg aggtggccat caccatcacc atcactgatg accggtt 957
<210> 23
<211> 398
<212> DNA
<213>West nile virus
<220>
<221> misc_feature
<223>The DNA sequence dna of EDIII albumen from WN viruses
<400> 23
taggcgcgcc aggaggtggc gggtctcagt tgaagggaac aacctatggc gtctgttcaa 60
aggctttcaa gtttcttggg actcccgcag acacaggtca cggcactgtg gtgttggaat 120
tgcagtacac tggcacggat ggaccttgca aagttcctat ctcgtcagtg gcttcattga 180
acgacctaac gccagtgggc agattggtca ctgtcaaccc ttttgtttca gtggccacgg 240
ccaacgctaa ggtcctgatt gaattggaac caccctttgg agactcatac atagtggtgg 300
gcagaggaga acaacagatc aatcaccatt ggcacaagtc tggaagcagc attggcaaag 360
gaggtggcca tcaccatcac catcactgat gaccggtt 398
<210> 24
<211> 398
<212> DNA
<213>Usutu virus isolated strains USU629-05
<220>
<221> misc_feature
<223>The DNA sequence dna of ED III albumen from USU viruses
<400> 24
taggcgcgcc aggaggtggc gggtctacac taaaaggcac cacctacggc atgtgcacgg 60
aaaagttttc ttttgcaaaa aatccggctg acacgggtca cggcactgtg gtccttgaac 120
tgcagtacac gggatctgac ggaccttgca aaatcccaat ttccattgtg gcatcacttt 180
ccgatctcac ccccattggt agaatggtta cagcaaaccc ttatgtggct tcatccgaag 240
ccaacgcgaa agtgttggtt gagatggaac caccatttgg agattcatac attgtggttg 300
gaagagggga taagcagata aaccatcact ggcacaaagc aggaagttcc attggaaaag 360
gtggaggcca ccatcaccat caccattgat gaccggtt 398
<210> 25
<211> 410
<212> DNA
<213>Japanese encephalitis virus strain GP05
<220>
<221> misc_feature
<223>The DNA sequence dna of ED III albumen from JE viruses
<400> 25
taggcgcgcc agggtccctg gagggaggtg gcgggtctgc tctgaaaggc acaacctatg 60
gcatgtgtac agaaaaattc tcgttcgcga aaaatccggc ggacactggt cacggaacag 120
ttgtcattga actctcctac tctgggagtg atggcccctg caaaattccg attgtctccg 180
tcgcgagcct caatgacatg actcctgttg ggcggctggt gacagtgaac ccctttgtcg 240
cggcttccag tgccaactca aaggtgctgg tcgagatgga accccccttc ggagactcct 300
atatcgtggt tggaagggga gacaagcaga tcaaccacca ttggcacaga gctggaagca 360
cgctgggcaa gggaggtggc catcaccatc accatcactg atgaccggtt 410
<210> 26
<211> 398
<212> DNA
<213>Kumlinge plants of A 52 of tick-brone encephalitis virus
<220>
<221> misc_feature
<223>The DNA sequence dna of ED III albumen from TBE viruses
<400> 26
taggcgcgcc agggtccctg gagggaggtg gcgggtctct ggaaaaactg aagatgaaag 60
gtcttacgta cacaatgtgt gacaaaacaa agttcacatg gaagagagct ccaacagata 120
gtgggcatga cacagtggtc atggaagtca cattctctgg aacaaagccc tgtaggatcc 180
cagtcagggc agtggcacat ggatctccag atgtgaacgt ggccatgctg ataacgccaa 240
accctacaat tgaaaacaat ggaggtggct tcatagagat gcagctgccc ccaggggata 300
acatcatcta tgttggggaa ctgagtcacc aatggttcca aaaagggagc agcattggag 360
gaggtggcca tcaccatcac catcactgat gaccggtt 398
<210> 27
<211> 404
<212> DNA
<213>1 type dengue virus strain FGA/NA d1d
<220>
<221> misc_feature
<223>The DNA sequence dna of ED III albumen from DEN-1 viruses
<400> 27
taggcgcgcc agggtccctg gagggaggtg gcgggtctct gactttaaaa gggatgtcat 60
atgtgatgtg cacaggctca tttaagctag agaaggaagt ggctgagacc cagcatggaa 120
ctgtcctagt gcaggttaaa tacgaaggaa cagatgcgcc atgcaagatc cccttttcga 180
cccaagatga gaaaggagtg acccagaatg ggagattgat aacagccaat cccatagtta 240
ctgacaaaga aaaaccaatc aacattgaga cagaaccacc ttttggtgag agctacatca 300
tagtaggggc aggtgaaaaa gctttgaaac taagctggtt caagaaagga agcagcatag 360
ggaaaggagg tggccatcac catcaccatc actgatgacc ggtt 404
<210> 28
<211> 404
<212> DNA
<213>Dengue fever virus 2
<220>
<221> misc_feature
<223>The DNA sequence dna of ED III albumen from DEN-2 viruses
<400> 28
taggcgcgcc agggtccctg gagggaggtg gcgggtctct acagctcaaa ggaatgtcat 60
attctatgtg tacaggaaag tttaaagttg tgaaggaaat agcagaaaca caacatggaa 120
caatagttct cagagtacaa tatgaagggg acggttctcc gtgcaagatc ccttttgaaa 180
taatggattt ggaaaaaaga catgtcttag gtcgcttgat cacagtcaac ccaattgtta 240
cagaaataga cagcccagtc aacatagaag cagaacctcc attcggagac agctacatca 300
ttataggagt agaaccggga caactgaagc tcagctggtt taagaaagga agttccattg 360
gccaaggagg tggccatcac catcaccatc actgatgacc ggtt 404
<210> 29
<211> 401
<212> DNA
<213>Dengue fever virus 3
<220>
<221> misc_feature
<223>The DNA sequence dna of ED III albumen from DEN-3 viruses
<400> 29
taggcgcgcc agggtccctg gagggaggtg gcgggtctga actcaagggg atgagctatg 60
caatgtgctt gaataccttt gtgttgaaga aagaagtctc cgaaacgcag catgggacaa 120
tactcattaa ggttgagtac aaaggggaag atgcaccttg caagattcct ttctccacag 180
aggatggaca agggaaagcc cacaatggta gactgatcac agccaaccca gtggttacta 240
agaaggagga gcctgtcaac attgaggctg aacctccttt tggggaaagc aacatagtga 300
ttggagttgg agacaaagcc ttgaaaatta actggtacaa gaagggaagc tcgattggga 360
agggaggtgg ccatcaccat caccatcact gatgaccggt t 401
<210> 30
<211> 401
<212> DNA
<213>Dengue fever virus 4
<220>
<221> misc_feature
<223>The DNA sequence dna of ED III albumen from DEN-4 viruses
<400> 30
taggcgcgcc agggtccctg gagggaggtg gcgggtctag aatcaaggga atgtcataca 60
cgatgtgctc aggaaagttc tcaattgaca aagagatggc agaaacacag catgggacaa 120
cagtggtgaa agtcaagtat gaaggtgctg gagctccgtg taaagtcccc atagagatac 180
gagatgtaaa taaggaaaaa gtggttgggc gtgtcatctc atccacccct ctagctgaga 240
ataccaacag tgtgaccaac atagaactgg aacccccctt tggggacagt tacatagtca 300
taggtgttgg gaacagtgca ttgacactcc attggttcag gaaaggaagt tctattggca 360
agggaggtgg ccatcaccat caccatcact gatgaccggt t 401
<210> 31
<211> 410
<212> DNA
<213>Yellow fever strain ASIBI
<220>
<221> misc_feature
<223>The DNA sequence dna of ED III albumen from YF Asibi viruses
<400> 31
taggcgcgcc agggtccctg gagggaggtg gcgggtcttc agctttgaca ctcaagggga 60
catcctacaa aatgtgcact gacaaaatgt cttttgtcaa gaacccaact gacactggcc 120
atggcactgt tgtgatgcag gtgaaagtgc caaaaggagc cccctgcaag attccagtga 180
tagtagctga tgatcttaca gcggcaatca ataaaggcat tttggttaca gttaacccca 240
tcgcctcaac caatgatgat gaagtgctga ttgaggtgaa cccacctttt ggagacagct 300
acattatcgt tgggacagga gattcacgtc tcacttacca gtggcacaaa gagggaagct 360
caataggaaa gggaggtggc catcaccatc accatcactg atgaccggtt 410
<210> 32
<211> 595
<212> DNA
<213>Artificial sequence
<220>
<223>Synthesize the DNA sequence dna of IFNAI genes
<400> 32
gcgcgccagg gtccctaggt ggcggatctg atgacgatga taaagatatc tgtgatctcc 60
ctgagaccca cagcctggat aacaggagga ccttgatgct cctggcacaa atgagcagaa 120
tctctccttc ctcctgtctg atggacagac atgactttgg atttccccag gaggagtttg 180
atggcaacca gttccagaag gctccagcca tctctgtcct ccatgagctg atccagcaga 240
tcttcaacct ctttaccaca aaagattcat ctgctgcttg ggatgaggac ctcctagaca 300
aattctgcac cgaactctac cagcagctga atgacttgga agcctgtgtg atgcaggagg 360
agagggtggg agaaactccc ctgatgaatg cggactccat cttggctgtg aagaaatact 420
tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc tgggaggttg 480
tcagagcaga aatcatgaga tccctctctt tatcaacaaa cttgcaagaa agattaagga 540
ggaaggaagg caagtggggc ggtggaagtc atcatcatca tcatcattga ccggt 595
<210> 33
<211> 469
<212> PRT
<213>Artificial sequence
<220>
<223>The amino acid sequence of fusion protein BiP-SNAP- intervening sequences-RVF-N+intervening sequence-(His) 6 label
<400> 33
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Gly Gly Gly Ser Asp Asn Tyr Gln Glu Leu Arg Val Gln
210 215 220
Phe Ala Ala Gln Ala Val Asp Arg Asn Glu Ile Glu Gln Trp Val Arg
225 230 235 240
Glu Phe Ala Tyr Gln Gly Phe Asp Ala Arg Arg Val Ile Glu Leu Leu
245 250 255
Lys Gln Tyr Gly Gly Ala Asp Trp Glu Lys Asp Ala Lys Lys Met Ile
260 265 270
Val Leu Ala Leu Thr Arg Gly Asn Lys Pro Arg Arg Met Met Met Lys
275 280 285
Met Ser Lys Glu Gly Lys Ala Thr Val Glu Ala Leu Ile Asn Lys Tyr
290 295 300
Lys Leu Lys Glu Gly Asn Pro Ser Arg Asp Glu Leu Thr Leu Ser Arg
305 310 315 320
Val Ala Ala Ala Leu Ala Gly Trp Thr Cys Gln Ala Leu Val Val Leu
325 330 335
Ser Glu Trp Leu Pro Val Thr Gly Thr Thr Met Asp Gly Leu Ser Pro
340 345 350
Ala Tyr Pro Arg His Met Met His Pro Ser Phe Ala Gly Met Val Asp
355 360 365
Pro Ser Leu Pro Gly Asp Tyr Leu Arg Ala Ile Leu Asp Ala His Ser
370 375 380
Leu Tyr Leu Leu Gln Phe Ser Arg Val Ile Asn Pro Asn Leu Arg Gly
385 390 395 400
Arg Thr Lys Glu Glu Val Ala Ala Thr Phe Thr Gln Pro Met Asn Ala
405 410 415
Ala Val Asn Ser Asn Phe Ile Ser His Glu Lys Arg Arg Glu Phe Leu
420 425 430
Lys Ala Phe Gly Leu Val Asp Ser Asn Gly Lys Pro Ser Ala Ala Val
435 440 445
Met Ala Ala Ala Gln Ala Tyr Lys Thr Ala Ala Gly Gly Gly Ser His
450 455 460
His His His His His
465
<210> 34
<211> 398
<212> PRT
<213>Artificial sequence
<220>
<223>The amino acid sequence of fusion protein BiP- SNAP- intervening sequences-IFNa+intervening sequence-(His) 6 label
Row
<400> 34
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln Ala
65 70 75 80
Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu Glu
85 90 95
Phe Pro Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser Phe Thr
100 105 110
Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly Glu Val
115 120 125
Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala Ala Thr
130 135 140
Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile Leu Ile
145 150 155 160
Pro Cys His Arg Val Ser Ser Ser Gly Ala Val Gly Gly Tyr Glu Gly
165 170 175
Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly His Arg Leu
180 185 190
Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro Gly Ser Leu
195 200 205
Gly Gly Gly Ser Asp Asp Asp Asp Lys Asp Ile Cys Asp Leu Pro Glu
210 215 220
Thr His Ser Leu Asp Asn Arg Arg Thr Leu Met Leu Leu Ala Gln Met
225 230 235 240
Ser Arg Ile Ser Pro Ser Ser Cys Leu Met Asp Arg His Asp Phe Gly
245 250 255
Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe Gln Lys Ala Pro Ala
260 265 270
Ile Ser Val Leu His Glu Leu Ile Gln Gln Ile Phe Asn Leu Phe Thr
275 280 285
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp Leu Leu Asp Lys Phe
290 295 300
Cys Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Met
305 310 315 320
Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met Asn Ala Asp Ser Ile
325 330 335
Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr Leu Tyr Leu Thr Glu
340 345 350
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met
355 360 365
Arg Ser Leu Ser Leu Ser Thr Asn Leu Gln Glu Arg Leu Arg Arg Lys
370 375 380
Glu Gly Lys Trp Gly Gly Gly Ser His His His His His His
385 390 395
<210> 35
<211> 333
<212> PRT
<213>Artificial sequence
<220>
<223>The amino acid sequence of fusion protein BiP-SNAP- intervening sequence-EDIII WN+ intervening sequences-(His) 6 label
Row
<400> 35
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Gly Gly Gly Ser Gln Leu Lys Gly Thr Thr Tyr Gly Val Cys Ser
210 215 220
Lys Ala Phe Lys Phe Leu Gly Thr Pro Ala Asp Thr Gly His Gly Thr
225 230 235 240
Val Val Leu Glu Leu Gln Tyr Thr Gly Thr Asp Gly Pro Cys Lys Val
245 250 255
Pro Ile Ser Ser Val Ala Ser Leu Asn Asp Leu Thr Pro Val Gly Arg
260 265 270
Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala Lys
275 280 285
Val Leu Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val
290 295 300
Gly Arg Gly Glu Gln Gln Ile Asn His His Trp His Lys Ser Gly Ser
305 310 315 320
Ser Ile Gly Lys Gly Gly Gly His His His His His His
325 330
<210> 36
<211> 335
<212> PRT
<213>Artificial sequence
<220>
<223>The amino of fusion protein BiP-SNAP- intervening sequences-EDIII DEN-1+intervening sequence-(His) 6 label
Acid sequence
<400> 36
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Glu Gly Gly Gly Gly Ser Leu Thr Leu Lys Gly Met Ser
210 215 220
Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys Glu Val Ala Glu
225 230 235 240
Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr Glu Gly Thr Asp
245 250 255
Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly Val Thr
260 265 270
Gln Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp Lys Glu
275 280 285
Lys Pro Ile Asn Ile Glu Thr Glu Pro Pro Phe Gly Glu Ser Tyr Ile
290 295 300
Ile Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser Trp Phe Lys Lys
305 310 315 320
Gly Ser Ser Ile Gly Lys Gly Gly Gly His His His His His His
325 330 335
<210> 37
<211> 335
<212> PRT
<213>Artificial sequence
<220>
<223>The amino of fusion protein BiP-SNAP- intervening sequences-EDIII DEN-2+intervening sequence-(His) 6 label
Acid sequence
<400> 37
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Glu Gly Gly Gly Gly Ser Leu Gln Leu Lys Gly Met Ser
210 215 220
Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile Ala Glu
225 230 235 240
Thr Gln His Gly Thr Ile Val Leu Arg Val Gln Tyr Glu Gly Asp Gly
245 250 255
Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys Arg His
260 265 270
Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu Ile Asp
275 280 285
Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile
290 295 300
Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Ser Trp Phe Lys Lys
305 310 315 320
Gly Ser Ser Ile Gly Gln Gly Gly Gly His His His His His His
325 330 335
<210> 38
<211> 334
<212> PRT
<213>Artificial sequence
<220>
<223>The amino of fusion protein BiP-SNAP- intervening sequences-EDIII DEN-3+intervening sequence-(His) 6 label
Acid sequence
<400> 38
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Glu Gly Gly Gly Gly Ser Glu Leu Lys Gly Met Ser Tyr
210 215 220
Ala Met Cys Leu Asn Thr Phe Val Leu Lys Lys Glu Val Ser Glu Thr
225 230 235 240
Gln His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu Asp Ala
245 250 255
Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys Ala His
260 265 270
Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys Glu Glu
275 280 285
Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn Ile Val
290 295 300
Ile Gly Val Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Lys Lys Gly
305 310 315 320
Ser Ser Ile Gly Lys Gly Gly Gly His His His His His His
325 330
<210> 39
<211> 334
<212> PRT
<213>Artificial sequence
<220>
<223>Fusion protein BiP-SNAP- intervening sequences-EDIII
The amino acid sequence of DEN-4+intervening sequence-(His) 6 label
<400> 39
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Glu Gly Gly Gly Gly Ser Arg Ile Lys Gly Met Ser Tyr
210 215 220
Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met Ala Glu Thr
225 230 235 240
Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly Ala Gly Ala
245 250 255
Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys Glu Lys Val
260 265 270
Val Gly Arg Val Ile Ser Ser Thr Pro Leu Ala Glu Asn Thr Asn Ser
275 280 285
Val Thr Asn Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val
290 295 300
Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg Lys Gly
305 310 315 320
Ser Ser Ile Gly Lys Gly Gly Gly His His His His His His
325 330
<210> 40
<211> 338
<212> PRT
<213>Artificial sequence
<220>
<223>Fusion protein BiP-SNAP- intervening sequences-EDIII
The amino acid sequence of JE+intervening sequence-(His) 6 label
<400> 40
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Glu Gly Gly Gly Gly Ser Ser Ala Leu Lys Gly Thr Thr
210 215 220
Tyr Gly Met Cys Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp
225 230 235 240
Thr Gly His Gly Thr Val Val Ile Glu Leu Ser Tyr Ser Gly Ser Asp
245 250 255
Gly Pro Cys Lys Ile Pro Ile Val Ser Val Ala Ser Leu Asn Asp Met
260 265 270
Thr Pro Val Gly Arg Leu Val Thr Val Asn Pro Phe Val Ala Ala Ser
275 280 285
Ser Ala Asn Ser Lys Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp
290 295 300
Ser Tyr Ile Val Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp
305 310 315 320
His Arg Ala Gly Ser Thr Leu Gly Lys Gly Gly Gly His His His His
325 330 335
His His
<210> 41
<211> 333
<212> PRT
<213>Artificial sequence
<220>
<223>Fusion protein BiP-SNAP- intervening sequences-EDIII
The amino acid sequence of TBE+intervening sequence-(His) 6 label
<400> 41
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Glu Gly Gly Gly Gly Ser Leu Glu Lys Leu Lys Met Lys
210 215 220
Gly Leu Thr Tyr Thr Met Cys Asp Lys Thr Lys Phe Thr Trp Lys Arg
225 230 235 240
Ala Pro Thr Asp Ser Gly His Asp Thr Val Val Met Glu Val Thr Phe
245 250 255
Ser Gly Thr Lys Pro Cys Arg Ile Pro Val Arg Ala Val Ala His Gly
260 265 270
Ser Pro Asp Val Asn Val Ala Met Leu Ile Thr Pro Asn Pro Thr Ile
275 280 285
Glu Asn Asn Gly Gly Gly Phe Ile Glu Met Gln Leu Pro Pro Gly Asp
290 295 300
Asn Ile Ile Tyr Val Gly Glu Leu Ser His Gln Trp Phe Gln Lys Gly
305 310 315 320
Ser Ser Ile Gly Gly Gly Gly His His His His His His
325 330
<210> 42
<211> 337
<212> PRT
<213>Artificial sequence
<220>
<223>Fusion protein BiP-SNAP- intervening sequences-EDIII
The amino acid sequence of USU+intervening sequence-(His) 6 label
<400> 42
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Glu Gly Gly Gly Gly Ser Thr Leu Lys Gly Thr Thr Tyr
210 215 220
Gly Met Cys Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp Thr
225 230 235 240
Gly His Gly Thr Val Val Leu Glu Leu Gln Tyr Thr Gly Ser Asp Gly
245 250 255
Pro Cys Lys Ile Pro Ile Ser Ile Val Ala Ser Leu Ser Asp Leu Thr
260 265 270
Pro Ile Gly Arg Met Val Thr Ala Asn Pro Tyr Val Ala Ser Ser Glu
275 280 285
Ala Asn Ala Lys Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp Ser
290 295 300
Tyr Ile Val Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp His
305 310 315 320
Lys Ala Gly Ser Ser Ile Gly Lys Gly Gly Gly His His His His His
325 330 335
His
<210> 43
<211> 337
<212> PRT
<213>Artificial sequence
<220>
<223>Fusion protein BiP-SNAP- intervening sequences-EDIII
The amino acid sequence of YF+intervening sequence-(His) 6 label
<400> 43
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Glu Gly Gly Gly Gly Ser Ser Ala Leu Thr Leu Lys Gly
210 215 220
Thr Ser Tyr Lys Met Cys Thr Asp Lys Met Ser Phe Val Lys Asn Pro
225 230 235 240
Thr Asp Thr Gly His Gly Thr Val Val Met Gln Val Lys Val Pro Lys
245 250 255
Gly Ala Pro Cys Lys Ile Pro Val Ile Val Ala Asp Asp Leu Thr Ala
260 265 270
Ala Ile Asn Lys Gly Ile Leu Val Thr Val Asn Pro Ile Ala Ser Thr
275 280 285
Asn Asp Asp Glu Val Leu Ile Glu Val Asn Pro Pro Phe Gly Asp Ser
290 295 300
Tyr Ile Ile Val Gly Thr Gly Asp Ser Arg Leu Thr Tyr Gln Trp His
305 310 315 320
Lys Glu Gly Ser Ser Ile Gly Lys Gly Gly Gly His His His His His
325 330 335
His
<210> 44
<211> 234
<212> PRT
<213>Artificial sequence
<220>
<223>Fusion protein:The amino acid sequence of BiP signal peptide+SNAP+ enterokinase site+EcoRV/XmaI+His labels
Row
<400> 44
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Gly Gly Gly Ser Asp Asp Asp Asp Lys Asp Ile Lys Asn
210 215 220
Pro Gly Gly Gly Ser His His His His His
225 230
<210> 45
<211> 211
<212> PRT
<213>Mouse
<400> 45
Met Ala Glu Thr Cys Lys Met Lys Tyr Ser Val Leu Asp Ser Pro Leu
1 5 10 15
Gly Lys Met Glu Leu Ser Gly Cys Glu Arg Gly Leu His Gly Ile Arg
20 25 30
Leu Leu Ser Gly Lys Thr Pro Asn Thr Asp Pro Thr Glu Ala Pro Ala
35 40 45
Thr Pro Glu Val Leu Gly Gly Pro Glu Gly Val Pro Glu Pro Leu Val
50 55 60
Gln Cys Thr Ala Trp Leu Glu Ala Tyr Phe Arg Glu Pro Ala Ala Thr
65 70 75 80
Glu Gly Leu Pro Leu Pro Ala Leu His His Pro Val Phe Gln Gln Asp
85 90 95
Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe
100 105 110
Gly Glu Thr Val Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro
115 120 125
Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Ser Asn Pro Val Pro
130 135 140
Ile Leu Ile Pro Cys His Arg Val Val Arg Ser Asp Gly Ala Ile Gly
145 150 155 160
His Tyr Ser Gly Gly Gly Gln Ala Val Lys Glu Trp Leu Leu Ala His
165 170 175
Glu Gly Ile Pro Thr Gly Gln Pro Ala Ser Lys Gly Leu Gly Leu Thr
180 185 190
Gly Thr Trp Leu Lys Ser Ser Phe Glu Ser Thr Ser Ser Glu Pro Ser
195 200 205
Gly Arg Asn
210
<210> 46
<211> 209
<212> PRT
<213>Rat
<400> 46
Met Ala Glu Ile Cys Lys Met Lys Tyr Thr Val Leu Asp Ser Pro Leu
1 5 10 15
Gly Lys Ile Glu Leu Ser Gly Cys Glu Arg Gly Leu His Gly Ile Arg
20 25 30
Phe Leu Ser Gly Lys Thr Pro Asn Thr Asp Pro Thr Glu Ala Pro Ala
35 40 45
Cys Pro Glu Val Leu Gly Gly Pro Glu Gly Val Pro Glu Pro Leu Val
50 55 60
Gln Cys Thr Ala Trp Leu Glu Ala Tyr Phe His Glu Pro Ala Ala Thr
65 70 75 80
Glu Gly Leu Pro Leu Pro Ala Leu His His Pro Val Phe Gln Gln Asp
85 90 95
Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe
100 105 110
Gly Glu Met Val Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro
115 120 125
Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Ser Asn Pro Val Pro
130 135 140
Ile Leu Ile Pro Cys His Arg Val Ile Arg Ser Asp Gly Ala Ile Gly
145 150 155 160
Asn Tyr Ser Gly Gly Gly Gln Thr Val Lys Glu Trp Leu Leu Ala His
165 170 175
Glu Gly Ile Pro Thr Gly Gln Pro Ala Ser Lys Gly Leu Gly Leu Ile
180 185 190
Gly Ser Trp Leu Lys Pro Ser Phe Glu Ser Ser Ser Pro Lys Pro Ser
195 200 205
Gly
<210> 47
<211> 555
<212> DNA
<213>Artificial sequence
<220>
<223>Encode SNAP DNA G/C low contents
<400> 47
attgaattta cagacaaaga ctgcgaaatg aaaagaacta cattggattc accacttggg 60
aagttggaac tgagtggatg cgagcaagga ttgcatgaaa ttaagcttct gggaaaagga 120
acttctgcag ctgatgcagt tgaagttcca gcaccagcag ctgttcttgg aggtcctgag 180
cccctcatgc aagccacagc ctggcttaac gcatatttcc accagcctga ggccattgag 240
gaatttccag tccccgccct tcaccatcct gtgtttcagc aggaaagctt cacccgccag 300
gtcctgtgga aattgctgaa ggtggtcaag tttggtgaag tgatttcata tcagcaactt 360
gctgcattgg ccggtaaccc cgcagctaca gctgccgtga aaactgctct cagcggaaat 420
cctgtgccca tcctgatccc ttgtcacaga gtcgtttcat cttccggagc tgtaggtggc 480
tatgaaggag gactggcagt taaggagtgg ctgctggctc atgaaggtca tagacttgga 540
aaacctggtt tggga 555
<210> 48
<211> 18
<212> PRT
<213>Artificial sequence
<220>
<223>BiP amino acid sequence
<400> 48
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly
<210> 49
<211> 738
<212> DNA
<213>TOSCANA viruses
<400> 49
atggacaact atcaagagct tcgagtccag tttgctgctc aagcagtgga ccgcaatgag 60
attgaacagt gggtccgaga gtttgcttat caagggtttg atgcccgtag agttatcgaa 120
ctcttaaagc agtatggtgg ggctgactgg gagaaggatg ccaagaaaat gattgttctg 180
gctctaactc gtggcaacaa gcccaggagg atgatgatga aaatgtcgaa agaaggcaaa 240
gcaactgtgg aggctctcat caacaagtat aagctaaagg aagggaatcc ttcccgggat 300
gagttgactc tatcacgagt tgctgccgcc ttggctggct ggacatgcca ggctttggtc 360
gtcttgagtg agtggcttcc tgtcactggg actaccatgg acggcctatc ccctgcatac 420
ccgaggcata tgatgcaccc cagctttgct ggcatggtgg atccttctct accaggagac 480
tatctaaggg caatattaga tgctcactct ctgtatctgc tgcagttctc ccgggtcatc 540
aacccaaacc tccgaggtag aacaaaagag gaggttgctg caacgttcac gcagccaatg 600
aatgcagcag tgaatagcaa ctttataagc catgagaaga ggagagaatt cttgaaagcc 660
tttggacttg tggattccaa tgggaagccg tcagctgctg tcatggcagc cgctcaggct 720
tacaagacag cagcctaa 738
<210> 50
<211> 66
<212> DNA
<213>Artificial sequence
<220>
<223>BiP- sample signals:For the extension insect BiP signal peptides of mammalian cell
<400> 50
atgaaactat gtattctact tgcagttgtt gcgttcgtag gattgtcctt acctacagct 60
ctggca 66
<210> 51
<211> 22
<212> PRT
<213>Artificial sequence
<220>
<223>BiP- sample signals:For the extension insect BiP signal peptides of mammalian cell
<400> 51
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala
20
<210> 52
<211> 21
<212> DNA
<213>Marmor erodens
<400> 52
gaaaacctgt acttccagag c 21
<210> 53
<211> 7
<212> PRT
<213>Marmor erodens
<400> 53
Glu Asn Leu Tyr Phe Gln Ser
1 5
<210> 54
<211> 699
<212> DNA
<213>People
<400> 54
gatatcatag gaggtcgaga agttattccc cactcacgcc cttacatggc atcacttcag 60
agaaatggtt cccacctatg cggtggtgta ctagttcacc caaagtgggt tctaacggca 120
gctcactgcc ttgcccagcg gatggctcag ctaaggcttg tacttggact tcacacccta 180
gacagccccg gtctcacctt ccacatcaag gcagctatcc agcaccctcg atacaagcca 240
gtacctgcac ttgagaacga cctagctcta cttcagctag acggtaaagt aaagcctagc 300
cggaccatcc gaccgttggc tctacctagt aagcgccagg tagttgcagc aggtactcgg 360
tgcagcatgg caggctgggg acttacccac cagggtggac gcctttcccg agtacttcgg 420
gagctagacc ttcaagtact ggacacccgc atgtgtaaca acagccgctt ttggaacgga 480
agcctatccc caagcatggt ttgcctagca gctgactcca aggaccaggc tccctgcaag 540
ggtgactcgg gtggacccct ggtttgtggc aaaggccggg tgttagccgg agttctttcc 600
ttcagctcca gggtatgcac tgacatcttc aagcctccag ttgcaaccgc tgttgcacct 660
tacgtttcct ggatcaggaa ggtcaccggt cgatcggcc 699
<210> 55
<211> 1400
<212> DNA
<213>Artificial sequence
<220>
<223>For the DNA sequence dna (Fig. 6) of insect BiP- samples/SNAP/ enterokinase/GrM/His labels of S2 cells
<400> 55
atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct cgggagatct 60
gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc ctctgggcaa gctggaactg 120
tctgggtgcg aacagggcct gcacgagatc aagctgctgg gcaaaggaac atctgccgcc 180
gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg gaccagagcc actgatgcag 240
gccaccgcct ggctcaacgc ctactttcac cagcctgagg ccatcgagga gttccctgtg 300
ccagccctgc accacccagt gttccagcag gagagcttta cccgccaggt gctgtggaaa 360
ctgctgaaag tggtgaagtt cggagaggtc atcagctacc agcagctggc cgccctggcc 420
ggcaatcccg ccgccaccgc cgccgtgaaa accgccctga gcggaaatcc cgtgcccatt 480
ctgatcccct gccaccgggt ggtgtctagc tctggcgccg tggggggcta cgagggcggg 540
ctcgccgtga aagagtggct gctggcccac gagggccaca gactgggcaa gcctgggctg 600
ggtcctgcag gtataggcgc gccagggtcc ctaggtggcg gatctgatga cgatgataaa 660
gatatcatag gaggtcgaga agttattccc cactcacgcc cttacatggc atcacttcag 720
agaaatggtt cccacctatg cggtggtgta ctagttcacc caaagtgggt tctaacggca 780
gctcactgcc ttgcccagcg gatggctcag ctaaggcttg tacttggact tcacacccta 840
gacagccccg gtctcacctt ccacatcaag gcagctatcc agcaccctcg atacaagcca 900
gtacctgcac ttgagaacga cctagctcta cttcagctag acggtaaagt aaagcctagc 960
cggaccatcc gaccgttggc tctacctagt aagcgccagg tagttgcagc aggtactcgg 1020
tgcagcatgg caggctgggg acttacccac cagggtggac gcctttcccg agtacttcgg 1080
gagctagacc ttcaagtact ggacacccgc atgtgtaaca acagccgctt ttggaacgga 1140
agcctatccc caagcatggt ttgcctagca gctgactcca aggaccaggc tccctgcaag 1200
ggtgactcgg gtggacccct ggtttgtggc aaaggccggg tgttagccgg agttctttcc 1260
ttcagctcca gggtatgcac tgacatcttc aagcctccag ttgcaaccgc tgttgcacct 1320
tacgtttcct ggatcaggaa ggtcaccggt cgatcggccc cgggcggtgg aagtcatcat 1380
catcatcatc attgaccggt 1400
<210> 56
<211> 434
<212> PRT
<213>Artificial sequence
<220>
<223>For the amino acid sequence (Fig. 6) of insect BiP- samples/SNAP/ enterokinase/GrM/His labels of S2 cells
<400> 56
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Gly Gly Gly Ser Asp Asp Asp Asp Lys Asp Ile Ile Gly
210 215 220
Gly Arg Glu Val Ile Pro His Ser Arg Pro Tyr Met Ala Ser Leu Gln
225 230 235 240
Arg Asn Gly Ser His Leu Cys Gly Gly Val Leu Val His Pro Lys Trp
245 250 255
Val Leu Thr Ala Ala His Cys Leu Ala Gln Arg Met Ala Gln Leu Arg
260 265 270
Leu Val Leu Gly Leu His Thr Leu Asp Ser Pro Gly Leu Thr Phe His
275 280 285
Ile Lys Ala Ala Ile Gln His Pro Arg Tyr Lys Pro Val Pro Ala Leu
290 295 300
Glu Asn Asp Leu Ala Leu Leu Gln Leu Asp Gly Lys Val Lys Pro Ser
305 310 315 320
Arg Thr Ile Arg Pro Leu Ala Leu Pro Ser Lys Arg Gln Val Val Ala
325 330 335
Ala Gly Thr Arg Cys Ser Met Ala Gly Trp Gly Leu Thr His Gln Gly
340 345 350
Gly Arg Leu Ser Arg Val Leu Arg Glu Leu Asp Leu Gln Val Leu Asp
355 360 365
Thr Arg Met Cys Asn Asn Ser Arg Phe Trp Asn Gly Ser Leu Ser Pro
370 375 380
Ser Met Val Cys Leu Ala Ala Asp Ser Lys Asp Gln Ala Pro Cys Lys
385 390 395 400
Gly Asp Ser Gly Gly Pro Leu Val Cys Gly Lys Gly Arg Val Leu Ala
405 410 415
Gly Val Leu Ser Phe Ser Ser Arg Val Cys Thr Asp Ile Phe Lys Pro
420 425 430
Pro Val
<210> 57
<211> 1243
<212> DNA
<213>Artificial sequence
<220>
<223>For BiP- samples/SNAP- samples/proTEV/IFN/His labels of mammalian cell such as HeLa cells
DNA sequence dna (Fig. 7)
<400> 57
gctagcacca tgaagttatg catattactg gccgtcgtgg cctttgttgg cctctcgctc 60
ccaacagctc tggcagacaa agactgcgaa atgaaaagaa ctacattgga ttcaccactt 120
gggaagttgg aactgagtgg atgcgagcaa ggattgcatg aaattaagct actgggaaaa 180
ggaacttctg ctgctgatgc agttgaagtt ccagcaccag cagctgttct tggaggtcct 240
gagcccctca tgcaagccac agcctggctt aacgcatatt tccaccagcc tgaggccatt 300
gaggaatttc cagtccccgc ccttcaccat cctgtgtttc agcaggagag cttcacccgc 360
caggtcctgt ggaaattgct gaaggtggtc aagtttggtg aagtgatttc atatcagcaa 420
cttgctgcat tggccggtaa ccccgcagct acagctgccg tgaaaactgc tctcagcgga 480
aatcctgtgc ccatcctgat cccttgtcac agagtcgttt catcttccgg agctgtaggt 540
ggctatgaag gaggactggc agttaaggag tggctgctgg ctcatgaagg tcatagactt 600
ggaaagcctg ggctgggtcc tgctggtata ggcgcgccag ggtccctagg tggcggatct 660
gaaaacctgt acttccagag cgatatctgt gatctccctg agacccacag cctggataac 720
aggaggacct tgatgctcct ggcacaaatg agcagaatct ctccttcctc ctgtctgatg 780
gacagacatg actttggatt tccccaggag gagtttgatg gcaaccagtt ccagaaggct 840
ccagccatct ctgtcctcca tgagctgatc cagcagattt tcaacctctt taccacaaaa 900
gattcatctg ctgcttggga tgaggacctc ctagacaaat tctgcaccga actctaccag 960
cagctgaatg acttggaagc ctgtgtgatg caggaggaga gggtgggaga aactcccctg 1020
atgaatgcgg actccatctt ggctgtgaag aaatacttcc gaagaatcac tctctatctg 1080
acagagaaga aatacagccc ttgtgcctgg gaggttgtca gagcagaaat catgagatcc 1140
ctctctttat caacaaactt gcaagaaaga ttaaggagga aggaaggcaa gtggcccggg 1200
ggtggaagtc atcatcatca tcatcattga agcttgcggc cgc 1243
<210> 58
<211> 406
<212> PRT
<213>Artificial sequence
<220>
<223>For the ammonia of BiP- samples/SNAP- samples/proTEV/IFN/His labels of mammalian cell such as HeLa cells
Base acid sequence (Fig. 7)
<400> 58
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Asp Lys Asp Cys Glu Met Lys Arg Thr Thr
20 25 30
Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly
35 40 45
Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala
50 55 60
Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu
65 70 75 80
Met Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala
85 90 95
Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln
100 105 110
Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys
115 120 125
Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn
130 135 140
Pro Ala Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val
145 150 155 160
Pro Ile Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val
165 170 175
Gly Gly Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His
180 185 190
Glu Gly His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly
195 200 205
Ala Pro Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Ser
210 215 220
Asp Ile Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr
225 230 235 240
Leu Met Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys Leu
245 250 255
Met Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn
260 265 270
Gln Phe Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu Ile Gln
275 280 285
Gln Ile Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp
290 295 300
Glu Asp Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln Leu Asn
305 310 315 320
Asp Leu Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro
325 330 335
Leu Met Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg
340 345 350
Ile Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
355 360 365
Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn Leu
370 375 380
Gln Glu Arg Leu Arg Arg Lys Glu Gly Lys Trp Pro Gly Gly Gly Ser
385 390 395 400
His His His His His His
405
<210> 59
<211> 798
<212> DNA
<213>Artificial sequence
<220>
<223> deSNAP Univ :BiP- samples/SNAP- samples/proTEV/MCS/
The DNA sequence dna (Fig. 8) of proTEV/His labels
<400> 59
gatcgcgagc tagcaccatg aaactatgta ttctacttgc agttgttgcg ttcgtaggat 60
tgtccttacc tacagctctg gcaagatctg acaaagactg cgaaatgaaa agaactacat 120
tggattcacc acttgggaag ttggaactga gtggatgcga gcaaggattg catgaaatta 180
agctactggg aaaaggaact tctgctgctg atgcagttga agttccagca ccagcagctg 240
ttcttggagg tcctgagccc ctcatgcaag ccacagcctg gcttaacgca tatttccacc 300
agcctgaggc cattgaggaa tttccagtcc ccgcccttca ccatcctgtg tttcagcagg 360
agagcttcac ccgccaggtc ctgtggaaat tgctgaaggt ggtcaagttt ggtgaagtga 420
tttcatatca gcaacttgct gcattggccg gtaaccccgc agctacagct gccgtgaaaa 480
ctgctctcag cggaaatcct gtgcccatcc tgatcccttg tcacagagtc gtttcatctt 540
ccggagctgt aggtggctat gaaggaggac tggcagttaa ggagtggctg ctggctcatg 600
aaggtcatag acttggaaag cctgggctgg gtcctgctgg tataggcgcg ccagggtccc 660
taggtggcgg atccgaaaac ctgtacttcc agagcgatat cggaggtgga ggcccgggag 720
agaatctata ttttcaaggg cccggcggag gtagtcacca tcatcaccat cactaatgac 780
cggtgcggcc gcaagctt 798
<210> 60
<211> 252
<212> PRT
<213>Artificial sequence
<220>
<223> deSNAP Univ :The amino acid of BiP- samples/SNAP- samples/proTEV/MCS/ proTEV/His sequence labels
Sequence (Fig. 8)
<400> 60
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Arg Ser Asp Lys Asp Cys Glu Met Lys Arg
20 25 30
Thr Thr Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu
35 40 45
Gln Gly Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala
50 55 60
Asp Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu
65 70 75 80
Pro Leu Met Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro
85 90 95
Glu Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe
100 105 110
Gln Gln Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val
115 120 125
Val Lys Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala
130 135 140
Gly Asn Pro Ala Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn
145 150 155 160
Pro Val Pro Ile Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly
165 170 175
Ala Val Gly Gly Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu
180 185 190
Ala His Glu Gly His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly
195 200 205
Ile Gly Ala Pro Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe
210 215 220
Gln Ser Asp Ile Gly Gly Gly Gly Pro Gly Glu Asn Leu Tyr Phe Gln
225 230 235 240
Gly Pro Gly Gly Gly Ser His His His His His His
245 250
<210> 61
<211> 5
<212> PRT
<213>Artificial sequence
<220>
<223>The amino acid cleavage site of film precursor prM from the dengue fever virus of serotype 1
<400> 61
Pro Thr Ala Leu Ala
1 5
<210> 62
<211> 6
<212> PRT
<213>Artificial sequence
<220>
<223>The amino acid cleavage site of enterokinase
<400> 62
Asp Asp Asp Asp Lys Asp
1 5
<210> 63
<211> 4
<212> PRT
<213>Artificial sequence
<220>
<223>Amino acid spacer sequence
<400> 63
Gly Gly Gly Ser
1
<210> 64
<211> 3461
<212> DNA
<213>Artificial sequence
<220>
<223>It is inserted into the DeSNAP- universal sequences of pUC57 plasmids
<400> 64
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga ttcgcgagct agcaccatga aactatgtat tctacttgca gttgttgcgt 480
tcgtaggatt gtccttacct acagctctgg caagatctga caaagactgc gaaatgaaaa 540
gaactacatt ggattcacca cttgggaagt tggaactgag tggatgcgag caaggattgc 600
atgaaattaa gctactggga aaaggaactt ctgctgctga tgcagttgaa gttccagcac 660
cagcagctgt tcttggaggt cctgagcccc tcatgcaagc cacagcctgg cttaacgcat 720
atttccacca gcctgaggcc attgaggaat ttccagtccc cgcccttcac catcctgtgt 780
ttcagcagga gagcttcacc cgccaggtcc tgtggaaatt gctgaaggtg gtcaagtttg 840
gtgaagtgat ttcatatcag caacttgctg cattggccgg taaccccgca gctacagctg 900
ccgtgaaaac tgctctcagc ggaaatcctg tgcccatcct gatcccttgt cacagagtcg 960
tttcatcttc cggagctgta ggtggctatg aaggaggact ggcagttaag gagtggctgc 1020
tggctcatga aggtcataga cttggaaagc ctgggctggg tcctgctggt ataggcgcgc 1080
cagggtccct aggtggcgga tccgaaaacc tgtacttcca gagcgatatc ggaggtggag 1140
gcccgggaga gaatctatat tttcaagggc ccggcggagg tagtcaccat catcaccatc 1200
actaatgacc ggtgcggccg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 1260
aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 1320
ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 1380
ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 1440
cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 1500
tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 1560
aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 1620
aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 1680
tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 1740
ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 1800
cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 1860
ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 1920
ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 1980
gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 2040
agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 2100
cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 2160
aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 2220
aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 2280
ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 2340
aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 2400
ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 2460
agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 2520
cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 2580
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 2640
gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 2700
cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 2760
cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 2820
ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 2880
catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 2940
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 3000
ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 3060
catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 3120
cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 3180
cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 3240
acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 3300
ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 3360
tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac 3420
attaacctat aaaaataggc gtatcacgag gccctttcgt c 3461
<210> 65
<211> 7
<212> PRT
<213>Marmor erodens
<400> 65
Glu Asn Leu Tyr Phe Gln Gly
1 5
<210> 66
<211> 21
<212> DNA
<213>Marmor erodens
<400> 66
gaaaacctgt acttccaggg g 21
<210> 67
<211> 530
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of SNAP- sample sequences
<400> 67
gacaaagact gcgaaatgaa aagaactaca ttggattcac cacttgggaa gttggaactg 60
agtggatgcg agcaaggatt gcatgaaatt aagctactgg gaaaaggaac ttctgctgct 120
gatgcagttg aagttccagc accagcagct gttcttggag gtcctgagcc cctcatgcaa 180
gccacagcct ggcttaacgc atatttccac cagcctgagg ccattgagga atttccagtc 240
cccgcccttc accatcctgt gtttcagcag gagagcttca cccgccaggt cctgtggaaa 300
ttgctgaagg tggtcaagtt tggtgaagtg atttcatatc agcaacttgc tgcattggcc 360
ggtaaccccg cagctacagc tgccgtgaaa actgctctca gcggaaatcc tgtgcccatc 420
ctgatccctt gtcacagagt cgtttcatct tccggagctg taggtggcta tgaaggagga 480
ctggcagtta aggagtggct gctggctcat gaaggtcata gacttggaaa 530
<210> 68
<211> 711
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of the MGMT sequences of modification
<400> 68
ctaggacaac ctgctccact agaacgattt gcttcacgac gtccacaggt ccttgcagta 60
cgtactgttt gtgatttagt acttggaaaa atggacaaag actgcgaaat gaaaagaact 120
acattggatt caccacttgg gaagttggaa ctgagtggat gcgagcaagg attgcatgaa 180
attaagctac tgggaaaagg aacttctgct gctgatgcag ttgaagttcc agcaccagca 240
gctgttcttg gaggtcctga gcccctcatg caatgtacag catggcttaa cgcatatttc 300
caccagcctg aggccattga ggaatttcca gtccccgccc ttcaccatcc tgtgtttcag 360
caggagagct tcacccgcca ggtcctgtgg aaattgctga aggtggtcaa gtttggtgaa 420
gtgatttcat atcagcaact tgctgcattg gccggtaacc ctaaagccgc gcgagcagtg 480
ggaggagcaa tgagaggcaa tcctgtgccc atcctgatcc cttgtcacag agtcgtttgt 540
tcttccggag ctgtaggcaa ctattctgga ggactggcag ttaaggagtg gctgctggct 600
catgaaggac atcgattagg caaaccaggt ttaggaggta gttcaggtct agcaggtgca 660
tggcttaagg gagcaggagc tacatctgga tcaccacctg ctggacgaaa t 711
<210> 69
<211> 900
<212> DNA
<213>Artificial sequence
<220>
<223>DeMGMT Univ DNA sequence dna (BIP samples/MGMT/proTEVx2/His labels) (Fig. 9)
<400> 69
atgaaactat gtattctact tgcagttgtt gcgttcgtag gattgtcctt acctacagct 60
ctggcaagat ctctaggaca acctgctcca ctagaacgat ttgcttcacg acgtccacag 120
gtccttgcag tacgtactgt ttgtgattta gtacttggaa aaatggacaa agactgcgaa 180
atgaaaagaa ctacattgga ttcaccactt gggaagttgg aactgagtgg atgcgagcaa 240
ggattgcatg aaattaagct actgggaaaa ggaacttctg ctgctgatgc agttgaagtt 300
ccagcaccag cagctgttct tggaggtcct gagcccctca tgcaatgtac agcatggctt 360
aacgcatatt tccaccagcc tgaggccatt gaggaatttc cagtccccgc ccttcaccat 420
cctgtgtttc agcaggagag cttcacccgc caggtcctgt ggaaattgct gaaggtggtc 480
aagtttggtg aagtgatttc atatcagcaa cttgctgcat tggccggtaa ccctaaagcc 540
gcgcgagcag tgggaggagc aatgagaggc aatcctgtgc ccatcctgat cccttgtcac 600
agagtcgttt gttcttccgg agctgtaggc aactattctg gaggactggc agttaaggag 660
tggctgctgg ctcatgaagg acatcgatta ggcaaaccag gtttaggagg tagttcaggt 720
ctagcaggtg catggcttaa gggagcagga gctacatctg gatcaccacc tgctggacga 780
aatggtggcg gatccgaaaa cctgtacttc cagagcgata tcggaggtgg aggcccggga 840
gagaatctat attttcaagg gcccggcgga ggtagtcacc atcatcacca tcactaatga 900
<210> 70
<211> 297
<212> PRT
<213>Artificial sequence
<220>
<223>DeMGMT Univ (BIP samples/MGMT/proTEVx2/His labels) amino acid sequence (Fig. 9)
<400> 70
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Arg Ser Leu Gly Gln Pro Ala Pro Leu Glu
20 25 30
Arg Phe Ala Ser Arg Arg Pro Gln Val Leu Ala Val Arg Thr Val Cys
35 40 45
Asp Leu Val Leu Gly Lys Met Asp Lys Asp Cys Glu Met Lys Arg Thr
50 55 60
Thr Leu Asp Ser Pro Leu Gly Lys Leu Glu Ser Gly Cys Glu Gln Gly
65 70 75 80
Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala
85 90 95
Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu
100 105 110
Met Gln Cys Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala
115 120 125
Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln
130 135 140
Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys
145 150 155 160
Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn
165 170 175
Pro Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Gly Asn Pro Val
180 185 190
Pro Ile Leu Ile Pro Cys His Arg Val Val Cys Ser Ser Gly Ala Val
195 200 205
Gly Asn Tyr Ser Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His
210 215 220
Glu Gly His Arg Leu Gly Lys Pro Gly Leu Gly Gly Ser Ser Gly Leu
225 230 235 240
Ala Gly Ala Trp Leu Lys Gly Ala Gly Ala Thr Ser Gly Ser Pro Pro
245 250 255
Ala Gly Arg Asn Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Ser Asp
260 265 270
Ile Gly Gly Gly Gly Pro Gly Glu Asn Leu Tyr Phe Gln Gly Pro Gly
275 280 285
Gly Gly Ser His His His His His His
290 295
<210> 71
<211> 3581
<212> DNA
<213>Artificial sequence
<220>
<223>Shuttle vector pUC57/DeMGMT DNA sequence dna (is inserted between Nru I and the Hind III from pUC57
DeMGMT sequences)
<400> 71
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgag 420
ctagcaccat gaaactatgt attctacttg cagttgttgc gttcgtagga ttgtccttac 480
ctacagctct ggcaagatct ctaggacaac ctgctccact agaacgattt gcttcacgac 540
gtccacaggt ccttgcagta cgtactgttt gtgatttagt acttggaaaa atggacaaag 600
actgcgaaat gaaaagaact acattggatt caccacttgg gaagttggaa ctgagtggat 660
gcgagcaagg attgcatgaa attaagctac tgggaaaagg aacttctgct gctgatgcag 720
ttgaagttcc agcaccagca gctgttcttg gaggtcctga gcccctcatg caatgtacag 780
catggcttaa cgcatatttc caccagcctg aggccattga ggaatttcca gtccccgccc 840
ttcaccatcc tgtgtttcag caggagagct tcacccgcca ggtcctgtgg aaattgctga 900
aggtggtcaa gtttggtgaa gtgatttcat atcagcaact tgctgcattg gccggtaacc 960
ctaaagccgc gcgagcagtg ggaggagcaa tgagaggcaa tcctgtgccc atcctgatcc 1020
cttgtcacag agtcgtttgt tcttccggag ctgtaggcaa ctattctgga ggactggcag 1080
ttaaggagtg gctgctggct catgaaggac atcgattagg caaaccaggt ttaggaggta 1140
gttcaggtct agcaggtgca tggcttaagg gagcaggagc tacatctgga tcaccacctg 1200
ctggacgaaa tggtggcgga tccgaaaacc tgtacttcca gagcgatatc ggaggtggag 1260
gcccgggaga gaatctatat tttcaagggc ccggcggagg tagtcaccat catcaccatc 1320
actaatgacc ggtgcggccg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 1380
aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 1440
ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 1500
ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 1560
cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 1620
tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 1680
aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 1740
aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 1800
tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 1860
ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 1920
cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 1980
ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 2040
ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 2100
gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 2160
agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 2220
cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 2280
aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 2340
aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 2400
ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 2460
aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 2520
ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 2580
agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 2640
cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 2700
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 2760
gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 2820
cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 2880
cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 2940
ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 3000
catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 3060
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 3120
ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 3180
catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 3240
cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 3300
cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 3360
acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 3420
ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 3480
tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac 3540
attaacctat aaaaataggc gtatcacgag gccctttcgt c 3581
<210> 72
<211> 1285
<212> DNA
<213>Artificial sequence
<220>
<223>DeSNAP Univ+IFN DNA sequence dna
<400> 72
tcgcgagcta gcaccatgaa actatgtatt ctacttgcag ttgttgcgtt cgtaggattg 60
tccttaccta cagctctggc aagatctgac aaagactgcg aaatgaaaag aactacattg 120
gattcaccac ttgggaagtt ggaactgagt ggatgcgagc aaggattgca tgaaattaag 180
ctactgggaa aaggaacttc tgctgctgat gcagttgaag ttccagcacc agcagctgtt 240
cttggaggtc ctgagcccct catgcaagcc acagcctggc ttaacgcata tttccaccag 300
cctgaggcca ttgaggaatt tccagtcccc gcccttcacc atcctgtgtt tcagcaggag 360
agcttcaccc gccaggtcct gtggaaattg ctgaaggtgg tcaagtttgg tgaagtgatt 420
tcatatcagc aacttgctgc attggccggt aaccccgcag ctacagctgc cgtgaaaact 480
gctctcagcg gaaatcctgt gcccatcctg atcccttgtc acagagtcgt ttcatcttcc 540
ggagctgtag gtggctatga aggaggactg gcagttaagg agtggctgct ggctcatgaa 600
ggtcatagac ttggaaagcc tgggctgggt cctgctggta taggcgcgcc agggtcccta 660
ggtggcggat ccgaaaacct gtacttccag agcgatatct gtgatctccc tgagacccac 720
agcctggata acaggaggac cttgatgctc ctggcacaaa tgagcagaat ctctccttcc 780
tcctgtctga tggacagaca tgactttgga tttccccagg aggagtttga tggcaaccag 840
ttccagaagg ctccagccat ctctgtcctc catgagctga tccagcagat tttcaacctc 900
tttaccacaa aagattcatc tgctgcttgg gatgaggacc tcctagacaa attctgcacc 960
gaactctacc agcagctgaa tgacttggaa gcctgtgtga tgcaggagga gagggtggga 1020
gaaactcccc tgatgaatgc ggactccatc ttggctgtga agaaatactt ccgaagaatc 1080
actctctatc tgacagagaa gaaatacagc ccttgtgcct gggaggttgt cagagcagaa 1140
atcatgagat ccctctcttt atcaacaaac ttgcaagaaa gattaaggag gaaggaaggc 1200
ccgggagaga atctatattt tcaagggccc ggcggaggta gtcaccatca tcaccatcac 1260
taatgaccgg tgcggccgca agctt 1285
<210> 73
<211> 414
<212> PRT
<213>Artificial sequence
<220>
<223>De SNAP Univ+IFN amino acid sequence
<400> 73
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Arg Ser Asp Lys Asp Cys Glu Met Lys Arg
20 25 30
Thr Thr Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu
35 40 45
Gln Gly Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Asp
50 55 60
Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro
65 70 75 80
Leu Met Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu
85 90 95
Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln
100 105 110
Gln Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val
115 120 125
Lys Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly
130 135 140
Asn Pro Ala Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro
145 150 155 160
Val Pro Ile Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala
165 170 175
Val Gly Gly Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala
180 185 190
His Glu Gly His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile
195 200 205
Gly Ala Pro Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln
210 215 220
Ser Asp Ile Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg
225 230 235 240
Thr Leu Met Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys
245 250 255
Leu Met Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly
260 265 270
Asn Gln Phe Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu Ile
275 280 285
Gln Gln Ile Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp
290 295 300
Asp Glu Asp Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln Leu
305 310 315 320
Asn Asp Leu Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr
325 330 335
Pro Leu Met Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg
340 345 350
Arg Ile Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp
355 360 365
Glu Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn
370 375 380
Leu Gln Glu Arg Leu Arg Arg Lys Glu Gly Pro Gly Glu Asn Leu Tyr
385 390 395 400
Phe Gln Gly Pro Gly Gly Gly Ser His His His His His His
405 410
<210> 74
<211> 1423
<212> DNA
<213>Artificial sequence
<220>
<223>DeMGMT Univ+IFN DNA sequence dna
<400> 74
tcgcgagcta gcaccatgaa actatgtatt ctacttgcag ttgttgcgtt cgtaggattg 60
tccttaccta cagctctggc aagatctcta ggacaacctg ctccactaga acgatttgct 120
tcacgacgtc cacaggtcct tgcagtacgt actgtttgtg atttagtact tggaaaaatg 180
gacaaagact gcgaaatgaa aagaactaca ttggattcac cacttgggaa gttggaactg 240
agtggatgcg agcaaggatt gcatgaaatt aagctactgg gaaaaggaac ttctgctgct 300
gatgcagttg aagttccagc accagcagct gttcttggag gtcctgagcc cctcatgcaa 360
tgtacagcat ggcttaacgc atatttccac cagcctgagg ccattgagga atttccagtc 420
cccgcccttc accatcctgt gtttcagcag gagagcttca cccgccaggt cctgtggaaa 480
ttgctgaagg tggtcaagtt tggtgaagtg atttcatatc agcaacttgc tgcattggcc 540
ggtaacccta aagccgcgcg agcagtggga ggagcaatga gaggcaatcc tgtgcccatc 600
ctgatccctt gtcacagagt cgtttgttct tccggagctg taggcaacta ttctggagga 660
ctggcagtta aggagtggct gctggctcat gaaggacatc gattaggcaa accaggttta 720
ggaggtagtt caggtctagc aggtgcatgg cttaagggag caggagctac atctggatca 780
ccacctgctg gacgaaatgg tggcggatcc gaaaacctgt acttccagag cgatatctgt 840
gatctccctg agacccacag cctggataac aggaggacct tgatgctcct ggcacaaatg 900
agcagaatct ctccttcctc ctgtctgatg gacagacatg actttggatt tccccaggag 960
gagtttgatg gcaaccagtt ccagaaggct ccagccatct ctgtcctcca tgagctgatc 1020
cagcagattt tcaacctctt taccacaaaa gattcatctg ctgcttggga tgaggacctc 1080
ctagacaaat tctgcaccga actctaccag cagctgaatg acttggaagc ctgtgtgatg 1140
caggaggaga gggtgggaga aactcccctg atgaatgcgg actccatctt ggctgtgaag 1200
aaatacttcc gaagaatcac tctctatctg acagagaaga aatacagccc ttgtgcctgg 1260
gaggttgtca gagcagaaat catgagatcc ctctctttat caacaaactt gcaagaaaga 1320
ttaaggagga aggaaggccc gggagagaat ctatattttc aagggcccgg cggaggtagt 1380
caccatcatc accatcacta atgaccggtg cggccgcaag ctt 1423
<210> 75
<211> 415
<212> PRT
<213>Artificial sequence
<220>
<223>DeMGMT Univ+IFN amino acid sequence
<400> 75
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Arg Ser Asp Lys Asp Cys Glu Met Lys Arg
20 25 30
Thr Thr Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu
35 40 45
Gln Gly Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala
50 55 60
Asp Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu
65 70 75 80
Pro Leu Met Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro
85 90 95
Glu Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe
100 105 110
Gln Gln Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val
115 120 125
Val Lys Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala
130 135 140
Gly Asn Pro Ala Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn
145 150 155 160
Pro Val Pro Ile Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly
165 170 175
Ala Val Gly Gly Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu
180 185 190
Ala His Glu Gly His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly
195 200 205
Ile Gly Ala Pro Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe
210 215 220
Gln Ser Asp Ile Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg
225 230 235 240
Arg Thr Leu Met Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser
245 250 255
Cys Leu Met Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp
260 265 270
Gly Asn Gln Phe Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu
275 280 285
Ile Gln Gln Ile Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala
290 295 300
Trp Asp Glu Asp Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln
305 310 315 320
Leu Asn Asp Leu Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu
325 330 335
Thr Pro Leu Met Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe
340 345 350
Arg Arg Ile Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
355 360 365
Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr
370 375 380
Asn Leu Gln Glu Arg Leu Arg Arg Lys Glu Gly Pro Gly Glu Asn Leu
385 390 395 400
Tyr Phe Gln Gly Pro Gly Gly Gly Ser His His His His His His
405 410 415
<210> 76
<211> 570
<212> PRT
<213>People
<400> 76
Gly Ala Thr Ala Thr Cys Ala Ala Cys Gly Gly Ala Gly Ala Cys Gly
1 5 10 15
Ala Cys Gly Cys Cys Thr Thr Thr Gly Cys Ala Ala Gly Gly Ala Gly
20 25 30
Ala Cys Cys Cys Ala Cys Gly Gly Thr Thr Gly Gly Thr Gly Cys Thr
35 40 45
Cys Ala Ala Ala Thr Ala Cys Cys Ala Gly Ala Gly Ala Ala Gly Ala
50 55 60
Thr Cys Cys Ala Ala Ala Ala Gly Gly Cys Cys Thr Thr Cys Gly Ala
65 70 75 80
Thr Gly Ala Thr Ala Thr Thr Gly Cys Cys Ala Ala Ala Thr Ala Cys
85 90 95
Thr Thr Cys Thr Cys Thr Ala Ala Gly Gly Ala Ala Gly Ala Gly Thr
100 105 110
Gly Gly Gly Ala Ala Ala Ala Gly Ala Thr Gly Ala Ala Ala Gly Cys
115 120 125
Cys Thr Cys Gly Gly Ala Gly Ala Ala Ala Ala Thr Cys Thr Thr Cys
130 135 140
Thr Ala Thr Gly Thr Gly Thr Ala Thr Ala Thr Gly Ala Ala Gly Ala
145 150 155 160
Gly Ala Ala Ala Gly Thr Ala Thr Gly Ala Gly Gly Cys Thr Ala Thr
165 170 175
Gly Ala Cys Thr Ala Ala Ala Cys Thr Ala Gly Gly Thr Thr Thr Cys
180 185 190
Ala Ala Gly Gly Cys Cys Ala Cys Cys Cys Thr Cys Cys Cys Ala Cys
195 200 205
Cys Thr Thr Thr Cys Ala Thr Gly Thr Gly Thr Ala Ala Thr Ala Ala
210 215 220
Ala Cys Gly Gly Gly Cys Cys Gly Ala Ala Gly Ala Cys Thr Thr Cys
225 230 235 240
Cys Ala Gly Gly Gly Gly Ala Ala Thr Gly Ala Thr Thr Thr Gly Gly
245 250 255
Ala Thr Ala Ala Thr Gly Ala Cys Cys Cys Thr Ala Ala Cys Cys Gly
260 265 270
Thr Gly Gly Gly Ala Ala Thr Cys Ala Gly Gly Thr Thr Gly Ala Ala
275 280 285
Cys Gly Thr Cys Cys Thr Cys Ala Gly Ala Thr Gly Ala Cys Thr Thr
290 295 300
Thr Cys Gly Gly Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly
305 310 315 320
Ala Ala Thr Cys Thr Cys Cys Cys Cys Gly Ala Ala Gly Ala Thr Cys
325 330 335
Ala Thr Gly Cys Cys Cys Ala Ala Gly Ala Ala Gly Cys Cys Ala Gly
340 345 350
Cys Ala Gly Ala Gly Gly Ala Ala Gly Gly Ala Ala Ala Thr Gly Ala
355 360 365
Thr Thr Cys Gly Gly Ala Gly Gly Ala Ala Gly Thr Gly Cys Cys Ala
370 375 380
Gly Ala Ala Gly Cys Ala Thr Cys Thr Gly Gly Cys Cys Cys Ala Cys
385 390 395 400
Ala Ala Ala Ala Thr Gly Ala Thr Gly Gly Gly Ala Ala Ala Gly Ala
405 410 415
Gly Cys Thr Gly Thr Gly Cys Cys Cys Thr Cys Cys Thr Gly Gly Ala
420 425 430
Ala Ala Ala Cys Cys Ala Ala Cys Thr Ala Cys Cys Thr Cys Thr Gly
435 440 445
Ala Gly Ala Ala Gly Ala Thr Thr Cys Ala Cys Gly Ala Gly Ala Gly
450 455 460
Ala Thr Cys Ala Gly Gly Ala Cys Cys Cys Ala Ala Ala Ala Gly Gly
465 470 475 480
Gly Gly Gly Gly Ala Ala Cys Ala Thr Gly Cys Cys Thr Gly Gly Ala
485 490 495
Cys Cys Cys Ala Cys Ala Gly Ala Cys Thr Gly Cys Gly Thr Gly Ala
500 505 510
Gly Ala Gly Ala Ala Ala Ala Cys Ala Gly Cys Thr Gly Gly Thr Gly
515 520 525
Ala Thr Thr Thr Ala Thr Gly Ala Ala Gly Ala Gly Ala Thr Cys Ala
530 535 540
Gly Cys Gly Ala Cys Cys Cys Thr Gly Ala Gly Gly Ala Ala Gly Ala
545 550 555 560
Thr Gly Ala Cys Gly Ala Gly Thr Ala Cys
565 570
<210> 77
<211> 1369
<212> DNA
<213>Artificial sequence
<220>
<223>DeSNAP Univ+SSX2 DNA sequence dna
<400> 77
tcgcgagcta gcaccatgaa actatgtatt ctacttgcag ttgttgcgtt cgtaggattg 60
tccttaccta cagctctggc aagatctgac aaagactgcg aaatgaaaag aactacattg 120
gattcaccac ttgggaagtt ggaactgagt ggatgcgagc aaggattgca tgaaattaag 180
ctactgggaa aaggaacttc tgctgctgat gcagttgaag ttccagcacc agcagctgtt 240
cttggaggtc ctgagcccct catgcaagcc acagcctggc ttaacgcata tttccaccag 300
cctgaggcca ttgaggaatt tccagtcccc gcccttcacc atcctgtgtt tcagcaggag 360
agcttcaccc gccaggtcct gtggaaattg ctgaaggtgg tcaagtttgg tgaagtgatt 420
tcatatcagc aacttgctgc attggccggt aaccccgcag ctacagctgc cgtgaaaact 480
gctctcagcg gaaatcctgt gcccatcctg atcccttgtc acagagtcgt ttcatcttcc 540
ggagctgtag gtggctatga aggaggactg gcagttaagg agtggctgct ggctcatgaa 600
ggtcatagac ttggaaagcc tgggctgggt cctgctggta taggcgcgcc agggtcccta 660
ggtggcggat ccgaaaacct gtacttccag agcgatatca acggagacga cgcctttgca 720
aggagaccca cggttggtgc tcaaatacca gagaagatcc aaaaggcctt cgatgatatt 780
gccaaatact tctctaagga agagtgggaa aagatgaaag cctcggagaa aatcttctat 840
gtgtatatga agagaaagta tgaggctatg actaaactag gtttcaaggc caccctccca 900
cctttcatgt gtaataaacg ggccgaagac ttccagggga atgatttgga taatgaccct 960
aaccgtggga atcaggttga acgtcctcag atgactttcg gcaggctcca gggaatctcc 1020
ccgaagatca tgcccaagaa gccagcagag gaaggaaatg attcggagga agtgccagaa 1080
gcatctggcc cacaaaatga tgggaaagag ctgtgccctc ctggaaaacc aactacctct 1140
gagaagattc acgagagatc aggacccaaa aggggggaac atgcctggac ccacagactg 1200
cgtgagagaa aacagctggt gatttatgaa gagatcagcg accctgagga agatgacgag 1260
tacgagaatc tatattttca aggcccggga gagaatctat attttcaagg gcccggcgga 1320
ggtagtcacc atcatcacca tcactaatga ccggtgcggc cgcaagctt 1369
<210> 78
<211> 415
<212> PRT
<213>Artificial sequence
<220>
<223>DeSNAP Univ+SSX2 amino acid sequence
<400> 78
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Arg Ser Asp Lys Asp Cys Glu Met Lys Arg
20 25 30
Thr Thr Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu
35 40 45
Gln Gly Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala
50 55 60
Asp Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu
65 70 75 80
Pro Leu Met Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro
85 90 95
Glu Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe
100 105 110
Gln Gln Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val
115 120 125
Val Lys Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala
130 135 140
Gly Asn Pro Ala Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn
145 150 155 160
Pro Val Pro Ile Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly
165 170 175
Ala Val Gly Gly Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu
180 185 190
Ala His Glu Gly His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly
195 200 205
Ile Gly Ala Pro Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe
210 215 220
Gln Ser Asp Ile Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg
225 230 235 240
Arg Thr Leu Met Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser
245 250 255
Cys Leu Met Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp
260 265 270
Gly Asn Gln Phe Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu
275 280 285
Ile Gln Gln Ile Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala
290 295 300
Trp Asp Glu Asp Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln
305 310 315 320
Leu Asn Asp Leu Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu
325 330 335
Thr Pro Leu Met Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe
340 345 350
Arg Arg Ile Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
355 360 365
Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr
370 375 380
Asn Leu Gln Glu Arg Leu Arg Arg Lys Glu Gly Pro Gly Glu Asn Leu
385 390 395 400
Tyr Phe Gln Gly Pro Gly Gly Gly Ser His His His His His His
405 410 415
<210> 79
<211> 1369
<212> DNA
<213>Artificial sequence
<220>
<223>DeMGMT Univ+SSX2 DNA sequence dna
<400> 79
tcgcgagcta gcaccatgaa actatgtatt ctacttgcag ttgttgcgtt cgtaggattg 60
tccttaccta cagctctggc aagatctgac aaagactgcg aaatgaaaag aactacattg 120
gattcaccac ttgggaagtt ggaactgagt ggatgcgagc aaggattgca tgaaattaag 180
ctactgggaa aaggaacttc tgctgctgat gcagttgaag ttccagcacc agcagctgtt 240
cttggaggtc ctgagcccct catgcaagcc acagcctggc ttaacgcata tttccaccag 300
cctgaggcca ttgaggaatt tccagtcccc gcccttcacc atcctgtgtt tcagcaggag 360
agcttcaccc gccaggtcct gtggaaattg ctgaaggtgg tcaagtttgg tgaagtgatt 420
tcatatcagc aacttgctgc attggccggt aaccccgcag ctacagctgc cgtgaaaact 480
gctctcagcg gaaatcctgt gcccatcctg atcccttgtc acagagtcgt ttcatcttcc 540
ggagctgtag gtggctatga aggaggactg gcagttaagg agtggctgct ggctcatgaa 600
ggtcatagac ttggaaagcc tgggctgggt cctgctggta taggcgcgcc agggtcccta 660
ggtggcggat ccgaaaacct gtacttccag agcgatatca acggagacga cgcctttgca 720
aggagaccca cggttggtgc tcaaatacca gagaagatcc aaaaggcctt cgatgatatt 780
gccaaatact tctctaagga agagtgggaa aagatgaaag cctcggagaa aatcttctat 840
gtgtatatga agagaaagta tgaggctatg actaaactag gtttcaaggc caccctccca 900
cctttcatgt gtaataaacg ggccgaagac ttccagggga atgatttgga taatgaccct 960
aaccgtggga atcaggttga acgtcctcag atgactttcg gcaggctcca gggaatctcc 1020
ccgaagatca tgcccaagaa gccagcagag gaaggaaatg attcggagga agtgccagaa 1080
gcatctggcc cacaaaatga tgggaaagag ctgtgccctc ctggaaaacc aactacctct 1140
gagaagattc acgagagatc aggacccaaa aggggggaac atgcctggac ccacagactg 1200
cgtgagagaa aacagctggt gatttatgaa gagatcagcg accctgagga agatgacgag 1260
tacgagaatc tatattttca aggcccggga gagaatctat attttcaagg gcccggcgga 1320
ggtagtcacc atcatcacca tcactaatga ccggtgcggc cgcaagctt 1369
<210> 80
<211> 489
<212> PRT
<213>Artificial sequence
<220>
<223>DeMGMT Univ+SSX2 amino acid sequence
<400> 80
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Arg Ser Leu Gly Gln Pro Ala Pro Leu Glu
20 25 30
Arg Phe Ala Ser Arg Arg Pro Gln Val Leu Ala Val Arg Thr Val Cys
35 40 45
Asp Leu Val Leu Gly Lys Met Asp Lys Asp Cys Glu Met Lys Arg Thr
50 55 60
Thr Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln
65 70 75 80
Gly Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp
85 90 95
Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro
100 105 110
Leu Met Gln Cys Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu
115 120 125
Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln
130 135 140
Gln Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val
145 150 155 160
Lys Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly
165 170 175
Asn Pro Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Gly Asn Pro
180 185 190
Val Pro Ile Leu Ile Pro Cys His Arg Val Val Cys Ser Ser Gly Ala
195 200 205
Val Gly Asn Tyr Ser Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala
210 215 220
His Glu Gly His Arg Leu Gly Lys Pro Gly Leu Gly Gly Ser Ser Gly
225 230 235 240
Leu Ala Gly Ala Trp Leu Lys Gly Ala Gly Ala Thr Ser Gly Ser Pro
245 250 255
Pro Ala Gly Arg Asn Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Ser
260 265 270
Asp Ile Asn Gly Asp Asp Ala Phe Ala Arg Arg Pro Thr Val Gly Ala
275 280 285
Gln Ile Pro Glu Lys Ile Gln Lys Ala Phe Asp Asp Ile Ala Lys Tyr
290 295 300
Phe Ser Lys Glu Glu Trp Glu Lys Met Lys Ala Ser Glu Lys Ile Phe
305 310 315 320
Tyr Val Tyr Met Lys Arg Lys Tyr Glu Ala Met Thr Lys Leu Gly Phe
325 330 335
Lys Ala Thr Leu Pro Pro Phe Met Cys Asn Lys Arg Ala Glu Asp Phe
340 345 350
Gln Gly Asn Asp Leu Asp Asn Asp Pro Asn Arg Gly Asn Gln Val Glu
355 360 365
Arg Pro Gln Met Thr Phe Gly Arg Leu Gln Gly Ile Ser Pro Lys Ile
370 375 380
Met Pro Lys Lys Pro Ala Glu Glu Gly Asn Asp Ser Glu Glu Val Pro
385 390 395 400
Glu Ala Ser Gly Pro Gln Asn Asp Gly Lys Glu Leu Cys Pro Pro Gly
405 410 415
Lys Pro Thr Thr Ser Glu Lys Ile His Glu Arg Ser Gly Pro Lys Arg
420 425 430
Gly Glu His Ala Trp Thr His Arg Leu Arg Glu Arg Lys Gln Leu Val
435 440 445
Ile Tyr Glu Glu Ile Ser Asp Pro Glu Glu Asp Asp Glu Tyr Glu Asn
450 455 460
Leu Tyr Phe Gln Gly Pro Gly Glu Asn Leu Tyr Phe Gln Gly Pro Gly
465 470 475 480
Gly Gly Ser His His His His His His
485
<210> 81
<211> 1299
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of ssBiP+SNAP+proTEV sites+SSX2+ proTEV+His labels
<400> 81
atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct cgggagatct 60
gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc ctctgggcaa gctggaactg 120
tctgggtgcg aacagggcct gcacgagatc aagctgctgg gcaaaggaac atctgccgcc 180
gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg gaccagagcc actgatgcag 240
gccaccgcct ggctcaacgc ctactttcac cagcctgagg ccatcgagga gttccctgtg 300
ccagccctgc accacccagt gttccagcag gagagcttta cccgccaggt gctgtggaaa 360
ctgctgaaag tggtgaagtt cggagaggtc atcagctacc agcagctggc cgccctggcc 420
ggcaatcccg ccgccaccgc cgccgtgaaa accgccctga gcggaaatcc cgtgcccatt 480
ctgatcccct gccaccgggt ggtgtctagc tctggcgccg tggggggcta cgagggcggg 540
ctcgccgtga aagagtggct gctggcccac gagggccaca gactgggcaa gcctgggctg 600
ggtcctgcag gtataggcgc gccagggtcc ctaggtggcg gatctgaaaa cctctacttc 660
cagagtgata tcaacggaga cgacgccttt gcaaggagac ccacggttgg tgctcaaata 720
ccagagaaga tccaaaaggc cttcgatgat attgccaaat acttctctaa ggaagagtgg 780
gaaaagatga aagcctcgga gaaaatcttc tatgtgtata tgaagagaaa gtatgaggct 840
atgactaaac taggtttcaa ggccaccctc ccacctttca tgtgtaataa acgggccgaa 900
gacttccagg ggaatgattt ggataatgac cctaaccgtg ggaatcaggt tgaacgtcct 960
cagatgactt tcggcaggct ccagggaatc tccccgaaga tcatgcccaa gaagccagca 1020
gaggaaggaa atgattcgga ggaagtgcca gaagcatctg gcccacaaaa tgatgggaaa 1080
gagctgtgcc ctcctggaaa accaactacc tctgagaaga ttcacgagag atcaggaccc 1140
aaaagggggg aacatgcctg gacccacaga ctgcgtgaga gaaaacagct ggtgatttat 1200
gaagagatca gcgaccctga ggaagatgac gagtacgaga atctatattt tcaaggcccg 1260
ggcggtggaa gtcaccatca tcaccatcac tgaccggta 1299
<210> 82
<211> 430
<212> PRT
<213>Artificial sequence
<220>
<223>SsBiP+SNAP+proTEV sites+SSX2+ proTEV+
The amino acid sequence of His labels
<400> 82
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Ser Asp Ile
210 215 220
Asn Gly Asp Asp Ala Phe Ala Arg Arg Pro Thr Val Gly Ala Gln Ile
225 230 235 240
Pro Glu Lys Ile Gln Lys Ala Phe Asp Asp Ile Ala Lys Tyr Phe Ser
245 250 255
Lys Glu Glu Trp Glu Lys Met Lys Ala Ser Glu Lys Ile Phe Tyr Val
260 265 270
Tyr Met Lys Arg Lys Tyr Glu Ala Met Thr Lys Leu Gly Phe Lys Ala
275 280 285
Thr Leu Pro Pro Phe Met Cys Asn Lys Arg Ala Glu Asp Phe Gln Gly
290 295 300
Asn Asp Leu Asp Asn Asp Pro Asn Arg Gly Asn Gln Val Glu Arg Pro
305 310 315 320
Gln Met Thr Phe Gly Arg Leu Gln Gly Ile Ser Pro Lys Ile Met Pro
325 330 335
Lys Lys Pro Ala Glu Glu Gly Asn Asp Ser Glu Glu Val Pro Glu Ala
340 345 350
Ser Gly Pro Gln Asn Asp Gly Lys Glu Leu Cys Pro Pro Gly Lys Pro
355 360 365
Thr Thr Ser Glu Lys Ile His Glu Arg Ser Gly Pro Lys Arg Gly Glu
370 375 380
His Ala Trp Thr His Arg Leu Arg Glu Arg Lys Gln Leu Val Ile Tyr
385 390 395 400
Glu Glu Ile Ser Asp Pro Glu Glu Asp Asp Glu Tyr Glu Asn Leu Tyr
405 410 415
Phe Gln Gly Pro Gly Gly Gly Ser His His His His His His
420 425 430
<210> 83
<211> 798
<212> DNA
<213>People
<400> 83
tctctagctg gagaaacagg tcaagaagct gcacctcttg atggagtact agcaaatcca 60
cctaatattt caagtctatc acctcgacaa cttcttggat ttccatgtgc agaagtatct 120
ggactaagta cagaacgtgt tcgagaacta gctgtagcat tagcacagaa aaatgtaaaa 180
ctatcaacag aacaacttcg atgtctagct catcgacttt ctgaaccacc tgaggatcta 240
gatgcacttc cattcgatct acttctattt ctaaatccag atgcattttc aggacctcaa 300
gcatgtactc gatttttttc tcgaattaca aaagcaaatg tcgatctact tccaagagga 360
gcaccagaac gacaacgact actacctgca gctctagcat gttggggagt acgaggatct 420
ctacttagtg aagcagatgt acgagctcta ggaggtctag cttgtgatct acctggacga 480
tttgtagcag aatctgcaga agtactacta ccacgacttg ttagttgtcc tggacctcta 540
gatcaagatc aacaagaagc tgctagagca gctcttcaag gtggtggacc tccttatgga 600
cctccatcaa catggtctgt atcaacaatg gatgcactac gaggacttct tcctgtacta 660
ggtcaaccta ttattcgaag tattccacaa ggtattgtag cagcatggcg acaacgatct 720
tctcgagatc catcttggcg acaacctgaa cgaactattc ttcgaccacg cccgggagag 780
aatctatatt ttcaaggg 798
<210> 84
<211> 1558
<212> DNA
<213>Artificial sequence
<220>
<223>DeSNAP Univ-NERCMSL DNA sequence dna
<400> 84
tcgcgagcta gcaccatgaa actatgtatt ctacttgcag ttgttgcgtt cgtaggattg 60
tccttaccta cagctctggc aagatctgac aaagactgcg aaatgaaaag aactacattg 120
gattcaccac ttgggaagtt ggaactgagt ggatgcgagc aaggattgca tgaaattaag 180
ctactgggaa aaggaacttc tgctgctgat gcagttgaag ttccagcacc agcagctgtt 240
cttggaggtc ctgagcccct catgcaagcc acagcctggc ttaacgcata tttccaccag 300
cctgaggcca ttgaggaatt tccagtcccc gcccttcacc atcctgtgtt tcagcaggag 360
agcttcaccc gccaggtcct gtggaaattg ctgaaggtgg tcaagtttgg tgaagtgatt 420
tcatatcagc aacttgctgc attggccggt aaccccgcag ctacagctgc cgtgaaaact 480
gctctcagcg gaaatcctgt gcccatcctg atcccttgtc acagagtcgt ttcatcttcc 540
ggagctgtag gtggctatga aggaggactg gcagttaagg agtggctgct ggctcatgaa 600
ggtcatagac ttggaaagcc tgggctgggt cctgctggta taggcgcgcc agggtcccta 660
ggtggcggat ccgaaaacct gtacttccag agcgatatca ggtctctagc tggagaaaca 720
ggtcaagaag ctgcacctct tgatggagta ctagcaaatc cacctaatat ttcaagtcta 780
tcacctcgac aacttcttgg atttccatgt gcagaagtat ctggactaag tacagaacgt 840
gttcgagaac tagctgtagc attagcacag aaaaatgtaa aactatcaac agaacaactt 900
cgatgtctag ctcatcgact ttctgaacca cctgaggatc tagatgcact tccattcgat 960
ctacttctat ttctaaatcc agatgcattt tcaggacctc aagcatgtac tcgatttttt 1020
tctcgaatta caaaagcaaa tgtcgatcta cttccaagag gagcaccaga acgacaacga 1080
ctactacctg cagctctagc atgttgggga gtacgaggat ctctacttag tgaagcagat 1140
gtacgagctc taggaggtct agcttgtgat ctacctggac gatttgtagc agaatctgca 1200
gaagtactac taccacgact tgttagttgt cctggacctc tagatcaaga tcaacaagaa 1260
gctgctagag cagctcttca aggtggtgga cctccttatg gacctccatc aacatggtct 1320
gtatcaacaa tggatgcact acgaggactt cttcctgtac taggtcaacc tattattcga 1380
agtattccac aaggtattgt agcagcatgg cgacaacgat cttctcgaga tccatcttgg 1440
cgacaacctg aacgaactat tcttcgacca cgcccgggag agaatctata ttttcaaggg 1500
cccggcggag gtagtcacca tcatcaccat cactaatgac cggtgcggcc gcaagctt 1558
<210> 85
<211> 504
<212> PRT
<213>Artificial sequence
<220>
<223>DeSNAP Univ-NERCMSL amino acid sequence
<400> 85
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Arg Ser Asp Lys Asp Cys Glu Met Lys Arg
20 25 30
Thr Thr Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu
35 40 45
Gln Gly Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala
50 55 60
Asp Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu
65 70 75 80
Pro Leu Met Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro
85 90 95
Glu Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe
100 105 110
Gln Gln Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val
115 120 125
Val Lys Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala
130 135 140
Gly Asn Pro Ala Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn
145 150 155 160
Pro Val Ile Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala
165 170 175
Val Gly Gly Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala
180 185 190
His Glu Gly His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile
195 200 205
Gly Ala Pro Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln
210 215 220
Ser Asp Ile Arg Ser Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro
225 230 235 240
Leu Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro
245 250 255
Arg Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr
260 265 270
Glu Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys
275 280 285
Leu Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro
290 295 300
Pro Glu Asp Leu Asp Ala Leu Pro Phe Asp Leu Leu Leu Phe Leu Asn
305 310 315 320
Pro Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg
325 330 335
Ile Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg
340 345 350
Gln Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser
355 360 365
Leu Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp
370 375 380
Leu Pro Gly Arg Phe Val Ala Glu Ser Ala Val Leu Leu Pro Arg Leu
385 390 395 400
Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg
405 410 415
Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp
420 425 430
Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly
435 440 445
Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg
450 455 460
Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile
465 470 475 480
Leu Arg Pro Arg Pro Gly Glu Asn Leu Tyr Phe Gln Gly Pro Gly Gly
485 490 495
Gly Ser His His His His His His
500
<210> 86
<211> 1558
<212> DNA
<213>Artificial sequence
<220>
<223>DeMGMT Univ-NERCMSL DNA sequence dna
<400> 86
tcgcgagcta gcaccatgaa actatgtatt ctacttgcag ttgttgcgtt cgtaggattg 60
tccttaccta cagctctggc aagatctgac aaagactgcg aaatgaaaag aactacattg 120
gattcaccac ttgggaagtt ggaactgagt ggatgcgagc aaggattgca tgaaattaag 180
ctactgggaa aaggaacttc tgctgctgat gcagttgaag ttccagcacc agcagctgtt 240
cttggaggtc ctgagcccct catgcaagcc acagcctggc ttaacgcata tttccaccag 300
cctgaggcca ttgaggaatt tccagtcccc gcccttcacc atcctgtgtt tcagcaggag 360
agcttcaccc gccaggtcct gtggaaattg ctgaaggtgg tcaagtttgg tgaagtgatt 420
tcatatcagc aacttgctgc attggccggt aaccccgcag ctacagctgc cgtgaaaact 480
gctctcagcg gaaatcctgt gcccatcctg atcccttgtc acagagtcgt ttcatcttcc 540
ggagctgtag gtggctatga aggaggactg gcagttaagg agtggctgct ggctcatgaa 600
ggtcatagac ttggaaagcc tgggctgggt cctgctggta taggcgcgcc agggtcccta 660
ggtggcggat ccgaaaacct gtacttccag agcgatatca ggtctctagc tggagaaaca 720
ggtcaagaag ctgcacctct tgatggagta ctagcaaatc cacctaatat ttcaagtcta 780
tcacctcgac aacttcttgg atttccatgt gcagaagtat ctggactaag tacagaacgt 840
gttcgagaac tagctgtagc attagcacag aaaaatgtaa aactatcaac agaacaactt 900
cgatgtctag ctcatcgact ttctgaacca cctgaggatc tagatgcact tccattcgat 960
ctacttctat ttctaaatcc agatgcattt tcaggacctc aagcatgtac tcgatttttt 1020
tctcgaatta caaaagcaaa tgtcgatcta cttccaagag gagcaccaga acgacaacga 1080
ctactacctg cagctctagc atgttgggga gtacgaggat ctctacttag tgaagcagat 1140
gtacgagctc taggaggtct agcttgtgat ctacctggac gatttgtagc agaatctgca 1200
gaagtactac taccacgact tgttagttgt cctggacctc tagatcaaga tcaacaagaa 1260
gctgctagag cagctcttca aggtggtgga cctccttatg gacctccatc aacatggtct 1320
gtatcaacaa tggatgcact acgaggactt cttcctgtac taggtcaacc tattattcga 1380
agtattccac aaggtattgt agcagcatgg cgacaacgat cttctcgaga tccatcttgg 1440
cgacaacctg aacgaactat tcttcgacca cgcccgggag agaatctata ttttcaaggg 1500
cccggcggag gtagtcacca tcatcaccat cactaatgac cggtgcggcc gcaagctt 1558
<210> 87
<211> 551
<212> PRT
<213>Artificial sequence
<220>
<223>DeMGMT Univ-NERCMSL amino acid sequence
<400> 87
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Arg Ser Leu Gly Gln Pro Ala Pro Leu Glu
20 25 30
Arg Phe Ala Ser Arg Arg Pro Gln Val Leu Ala Val Arg Thr Val Cys
35 40 45
Asp Leu Val Leu Gly Lys Met Asp Lys Asp Cys Glu Met Lys Arg Thr
50 55 60
Thr Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln
65 70 75 80
Gly Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp
85 90 95
Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro
100 105 110
Leu Met Gln Cys Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu
115 120 125
Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln
130 135 140
Gln Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val
145 150 155 160
Lys Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly
165 170 175
Asn Pro Lys Ala Ala Arg Ala Val Gly Gly Ala Met Arg Gly Asn Pro
180 185 190
Val Pro Ile Leu Ile Pro Cys His Arg Val Val Cys Ser Ser Gly Ala
195 200 205
Val Gly Asn Tyr Ser Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala
210 215 220
His Glu Gly His Arg Gly Lys Pro Gly Leu Gly Gly Ser Ser Gly Leu
225 230 235 240
Ala Gly Ala Trp Leu Lys Gly Ala Gly Ala Thr Ser Gly Ser Pro Pro
245 250 255
Ala Gly Arg Asn Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Ser Asp
260 265 270
Ile Arg Ser Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu Asp
275 280 285
Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg Gln
290 295 300
Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu Arg
305 310 315 320
Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu Ser
325 330 335
Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro Glu
340 345 350
Asp Leu Asp Ala Leu Pro Phe Asp Leu Leu Leu Phe Leu Asn Pro Asp
355 360 365
Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile Thr
370 375 380
Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln Arg
385 390 395 400
Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu Leu
405 410 415
Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu Pro
420 425 430
Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu Val
435 440 445
Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg Ala
450 455 460
Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp Ser
465 470 475 480
Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly Gln
485 490 495
Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg Gln
500 505 510
Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile Leu
515 520 525
Arg Pro Arg Pro Gly Glu Asn Leu Tyr Phe Gln Gly Pro Gly Gly Gly
530 535 540
Ser His His His His His His
545 550
<210> 88
<211> 543
<212> DNA
<213>People
<400> 88
gatatccagc tcttccacct acagaaggag ctggcagaac tccgagagtc taccagccag 60
atgcacacag catcatcttt ggagaagcaa ataggccacc ccagtccacc ccctgaaaaa 120
aaggagctga ggaaagtggc ccatttaaca ggcaagtcca actcaaggtc catgcctctg 180
gaatgggaag acacctatgg aattgtcctg ctttctggag tgaagtataa gaagggtggc 240
cttgtgatca atgaaactgg gctgtacttt gtatattcca aagtatactt ccggggtcaa 300
tcttgcaaca acctgcccct gagccacaag gtctacatga ggaactctaa gtatccccag 360
gatctggtga tgatggaggg gaagatgatg agctactgca ctactgggca gatgtgggcc 420
cgcagcagct acctgggggc agtgttcaat cttaccagtg ctgatcattt atatgtcaac 480
gtatctgagc tctctctggt caattttgag gaatctcaga cgtttttcgg cttatataag 540
ctc 543
<210> 89
<211> 1250
<212> DNA
<213>Artificial sequence
<220>
<223>SsBiP-SNAP-proTEV sites-sFasL DNA sequence dna
<400> 89
atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct cgggagatct 60
gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc ctctgggcaa gctggaactg 120
tctgggtgcg aacagggcct gcacgagatc aagctgctgg gcaaaggaac atctgccgcc 180
gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg gaccagagcc actgatgcag 240
gccaccgcct ggctcaacgc ctactttcac cagcctgagg ccatcgagga gttccctgtg 300
ccagccctgc accacccagt gttccagcag gagagcttta cccgccaggt gctgtggaaa 360
ctgctgaaag tggtgaagtt cggagaggtc atcagctacc agcagctggc cgccctggcc 420
ggcaatcccg ccgccaccgc cgccgtgaaa accgccctga gcggaaatcc cgtgcccatt 480
ctgatcccct gccaccgggt ggtgtctagc tctggcgccg tggggggcta cgagggcggg 540
ctcgccgtga aagagtggct gctggcccac gagggccaca gactgggcaa gcctgggctg 600
ggtcctgcag gtataggcgc gccagggtcc ctaggtggcg gatctgaaaa cctctacttc 660
cagagtgata tccagctctt ccacctacag aaggagctgg cagaactccg agagtctacc 720
agccagatgc acacagcatc atctttggag aagcaaatag gccaccccag tccaccccct 780
gaaaaaaagg agctgaggaa agtggcccat ttaacaggca agtccaactc aaggtccatg 840
cctctggaat gggaagacac ctatggaatt gtcctgcttt ctggagtgaa gtataagaag 900
ggtggccttg tgatcaatga aactgggctg tactttgtat attccaaagt atacttccgg 960
ggtcaatctt gcaacaacct gcccctgagc cacaaggtct acatgaggaa ctctaagtat 1020
ccccaggatc tggtgatgat ggaggggaag atgatgagct actgcactac tgggcagatg 1080
tgggcccgca gcagctacct gggggcagtg ttcaatctta ccagtgctga tcatttatat 1140
gtcaacgtat ctgagctctc tctggtcaat tttgaggaat ctcagacgtt tttcggctta 1200
tataagctcc cgggcggtgg aagtcatcat catcatcatc attgaccggt 1250
<210> 90
<211> 414
<212> PRT
<213>Artificial sequence
<220>
<223>SsBiP-SNAP-proTEV sites sFasL amino acid sequence
<400> 90
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
20 25 30
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
35 40 45
Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
50 55 60
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
65 70 75 80
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
85 90 95
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
100 105 110
Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly
115 120 125
Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro Ala
130 135 140
Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro Ile
145 150 155 160
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly
165 170 175
Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly
180 185 190
His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro
195 200 205
Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Ser Asp Ile
210 215 220
Gln Leu Phe His Leu Gln Lys Glu Leu Ala Glu Leu Arg Glu Ser Thr
225 230 235 240
Ser Gln Met His Thr Ala Ser Ser Leu Glu Lys Gln Ile Gly His Pro
245 250 255
Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg Lys Val Ala His Leu Thr
260 265 270
Gly Lys Ser Asn Ser Arg Ser Met Pro Leu Glu Trp Glu Asp Thr Tyr
275 280 285
Gly Ile Val Leu Leu Ser Gly Val Lys Tyr Lys Lys Gly Gly Leu Val
290 295 300
Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser Lys Val Tyr Phe Arg
305 310 315 320
Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser His Lys Val Tyr Met Arg
325 330 335
Asn Ser Lys Tyr Pro Gln Asp Leu Val Met Met Glu Gly Lys Met Met
340 345 350
Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala Arg Ser Ser Tyr Leu Gly
355 360 365
Ala Val Phe Asn Leu Thr Ser Ala Asp His Leu Tyr Val Asn Val Ser
370 375 380
Glu Leu Ser Leu Val Asn Phe Glu Glu Ser Gln Thr Phe Phe Gly Leu
385 390 395 400
Tyr Lys Leu Pro Gly Gly Gly Ser His His His His His His
405 410
<210> 91
<211> 2976
<212> DNA
<213>People
<220>
<221> misc_feature
<223>CNTN4 sequences, from amino acid/11 9 to 990
<400> 91
agatctatga ggttgccatg ggaactgctg gtactgcaat cattcatttt gtgccttgca 60
gatgattcca cactgcatgg cccgattttt attcaagaac caagtcctgt aatgttccct 120
ttggattctg aggagaaaaa agtgaagctc aattgtgaag ttaaaggaaa tccaaaacct 180
catatcaggt ggaagttaaa tggaacagat gttgacactg gtatggattt ccgctacagt 240
gttgttgaag ggagcttgtt gatcaataac cccaataaaa cccaagatgc tggaacgtac 300
cagtgcacag cgacaaactc gtttggaaca attgttagca gagaagcaaa gctgcagttt 360
gcttatcttg acaactttaa aacaagaaca agaagcactg tgtctgtccg tcgaggtcaa 420
ggaatggtgc tactgtgtgg cccgccaccc cattctggag agctgagtta tgcctggatc 480
ttcaatgaat acccttccta tcaggataat cgccgctttg tttctcaaga gactgggaat 540
ctgtatattg ccaaagtaga aaaatcagat gttgggaatt atacctgtgt ggttaccaat 600
accgtgacaa accacaaggt cctggggcca cctacaccac taatattgag aaatgatgga 660
gtgatgggtg aatatgagcc caaaatagaa gtgcagttcc cagaaacagt tccgactgca 720
aaaggagcaa cggtgaagct ggaatgcttt gctttaggaa atccagtacc aactattatc 780
tggcgaagag ctgatggaaa gccaatagca aggaaagcca gaagacacaa gtcaaatgga 840
attcttgaga tccctaattt tcagcaggag gatgctggtt tatatgaatg tgtagctgaa 900
aattccagag ggaaaaatgt agcaagggga cagctaactt tctatgctca acctaattgg 960
attcaaaaaa taaatgatat tcacgtggcc atggaagaaa atgtcttttg ggaatgtaaa 1020
gcaaatggaa ggcctaagcc tacatacaag tggctaaaaa atggcgaacc tctgctaact 1080
cgggatagaa ttcaaattga gcaaggaaca ctcaacataa caatagtgaa cctctcagat 1140
gctggcatgt atcagtgttt ggcagagaat aaacatggag ttatcttttc caacgcagag 1200
cttagtgtta tagctgtagg tccagatttt tcaagaacac tcttgaaaag agtaactctt 1260
gtcaaagtgg gaggtgaagt tgtcattgag tgtaagccaa aagcgtctcc aaaacctgtt 1320
tacacctgga agaaaggaag ggatatatta aaagaaaatg aaagaattac catttctgaa 1380
gatggaaacc tcagaatcat caacgttact aaatcagacg ctgggagtta tacctgtata 1440
gccactaacc attttggaac tgctagcagt actggaaact tggtagtgaa agatccaaca 1500
agggtaatgg tacccccttc cagtatggat gtcactgttg gagagagtat tgttttaccg 1560
tgccaggtaa cgcatgatca ctcgctagac atcgtgttta cttggtcatt taatggacac 1620
ctgatagact ttgacagaga tggggaccac tttgaaagag ttggagggca ggattcagct 1680
ggtgatttga tgatccgaaa catccaactg aagcatgctg ggaaatatgt ctgcatggtc 1740
caaacaagtg tggacaggct atctgctgct gcagacctga ttgtaagagg tcctccaggt 1800
cccccagagg ctgtgacaat agacgaaatc acagatacca ctgctcagct ctcctggaga 1860
cccggtcctg acaaccacag ccccatcacc atgtatgtca ttcaagccag gactccattc 1920
tccgtgggct ggcaagcagt cagtacagtc ccagaactca ttgatgggaa gacattcaca 1980
gcgaccgtgg tgggtttgaa cccttgggtt gaatatgaat tccgcacagt tgcagccaac 2040
gtgattggga ttggggagcc cagccgcccc tcagagaaac ggagaacaga agaagctctc 2100
cccgaagtca caccagcgaa tgtcagtggt ggcggaggca gcaaatctga actggttata 2160
acctgggaga cggtccctga ggaattacag aatggtcgag gctttggtta tgtggtggcc 2220
ttccggccct acggtaaaat gatctggatg ctgacagtgc tggcctcagc tgatgcctct 2280
agatacgtgt tcaggaatga gagcgtgcac cccttctctc cctttgaggt taaagtaggt 2340
gtcttcaaca acaaaggaga aggccctttc agtcccacca cggtggtgta ttctgcagaa 2400
gaagaaccca ccaaaccacc agccagtatc tttgccagaa gtctttctgc cacagatatt 2460
gaagttttct gggcctcccc actggagaag aatagaggac gaatacaagg ttatgaggtt 2520
aaatattgga gacatgaaga caaagaagaa aatgctagaa aaatacgaac agttggaaat 2580
cagacatcaa caaaaatcac gaacttaaaa ggcagtgtgc tgtatcactt agctgtcaag 2640
gcatataatt ctgctgggac aggcccctct agtgcaacag tcaatgtgac aacccgaaag 2700
ccaccaccaa gtcaaccccc cggaaacatc atatggaatt catcagactc caaaattatc 2760
ctgaattggg atcaagtgaa ggccctggat aatgagtcgg aagtaaaagg atacaaagtc 2820
ttgtacagat ggaacagaca aagcagcaca tctgtcattg aaacaaataa aacatcggtg 2880
gagctttctt tgcctttcga tgaagattat ataatagaaa ttaagccatt cagcgacgga 2940
ggagatggca gcagcagtga acaaattcga attccc 2976
<210> 92
<211> 3688
<212> DNA
<213>Artificial sequence
<220>
<223>The DNA sequence dna of BiP-CNTN419-990-SNAP-ProTEV-His labels
<400> 92
atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct cgggagatct 60
atgaggttgc catgggaact gctggtactg caatcattca ttttgtgcct tgcagatgat 120
tccacactgc atggcccgat ttttattcaa gaaccaagtc ctgtaatgtt ccctttggat 180
tctgaggaga aaaaagtgaa gctcaattgt gaagttaaag gaaatccaaa acctcatatc 240
aggtggaagt taaatggaac agatgttgac actggtatgg atttccgcta cagtgttgtt 300
gaagggagct tgttgatcaa taaccccaat aaaacccaag atgctggaac gtaccagtgc 360
acagcgacaa actcgtttgg aacaattgtt agcagagaag caaagctgca gtttgcttat 420
cttgacaact ttaaaacaag aacaagaagc actgtgtctg tccgtcgagg tcaaggaatg 480
gtgctactgt gtggcccgcc accccattct ggagagctga gttatgcctg gatcttcaat 540
gaataccctt cctatcagga taatcgccgc tttgtttctc aagagactgg gaatctgtat 600
attgccaaag tagaaaaatc agatgttggg aattatacct gtgtggttac caataccgtg 660
acaaaccaca aggtcctggg gccacctaca ccactaatat tgagaaatga tggagtgatg 720
ggtgaatatg agcccaaaat agaagtgcag ttcccagaaa cagttccgac tgcaaaagga 780
gcaacggtga agctggaatg ctttgcttta ggaaatccag taccaactat tatctggcga 840
agagctgatg gaaagccaat agcaaggaaa gccagaagac acaagtcaaa tggaattctt 900
gagatcccta attttcagca ggaggatgct ggtttatatg aatgtgtagc tgaaaattcc 960
agagggaaaa atgtagcaag gggacagcta actttctatg ctcaacctaa ttggattcaa 1020
aaaataaatg atattcacgt ggccatggaa gaaaatgtct tttgggaatg taaagcaaat 1080
ggaaggccta agcctacata caagtggcta aaaaatggcg aacctctgct aactcgggat 1140
agaattcaaa ttgagcaagg aacactcaac ataacaatag tgaacctctc agatgctggc 1200
atgtatcagt gtttggcaga gaataaacat ggagttatct tttccaacgc agagcttagt 1260
gttatagctg taggtccaga tttttcaaga acactcttga aaagagtaac tcttgtcaaa 1320
gtgggaggtg aagttgtcat tgagtgtaag ccaaaagcgt ctccaaaacc tgtttacacc 1380
tggaagaaag gaagggatat attaaaagaa aatgaaagaa ttaccatttc tgaagatgga 1440
aacctcagaa tcatcaacgt tactaaatca gacgctggga gttatacctg tatagccact 1500
aaccattttg gaactgctag cagtactgga aacttggtag tgaaagatcc aacaagggta 1560
atggtacccc cttccagtat ggatgtcact gttggagaga gtattgtttt accgtgccag 1620
gtaacgcatg atcactcgct agacatcgtg tttacttggt catttaatgg acacctgata 1680
gactttgaca gagatgggga ccactttgaa agagttggag ggcaggattc agctggtgat 1740
ttgatgatcc gaaacatcca actgaagcat gctgggaaat atgtctgcat ggtccaaaca 1800
agtgtggaca ggctatctgc tgctgcagac ctgattgtaa gaggtcctcc aggtccccca 1860
gaggctgtga caatagacga aatcacagat accactgctc agctctcctg gagacccggt 1920
cctgacaacc acagccccat caccatgtat gtcattcaag ccaggactcc attctccgtg 1980
ggctggcaag cagtcagtac agtcccagaa ctcattgatg ggaagacatt cacagcgacc 2040
gtggtgggtt tgaacccttg ggttgaatat gaattccgca cagttgcagc caacgtgatt 2100
gggattgggg agcccagccg cccctcagag aaacggagaa cagaagaagc tctccccgaa 2160
gtcacaccag cgaatgtcag tggtggcgga ggcagcaaat ctgaactggt tataacctgg 2220
gagacggtcc ctgaggaatt acagaatggt cgaggctttg gttatgtggt ggccttccgg 2280
ccctacggta aaatgatctg gatgctgaca gtgctggcct cagctgatgc ctctagatac 2340
gtgttcagga atgagagcgt gcaccccttc tctccctttg aggttaaagt aggtgtcttc 2400
aacaacaaag gagaaggccc tttcagtccc accacggtgg tgtattctgc agaagaagaa 2460
cccaccaaac caccagccag tatctttgcc agaagtcttt ctgccacaga tattgaagtt 2520
ttctgggcct ccccactgga gaagaataga ggacgaatac aaggttatga ggttaaatat 2580
tggagacatg aagacaaaga agaaaatgct agaaaaatac gaacagttgg aaatcagaca 2640
tcaacaaaaa tcacgaactt aaaaggcagt gtgctgtatc acttagctgt caaggcatat 2700
aattctgctg ggacaggccc ctctagtgca acagtcaatg tgacaacccg aaagccacca 2760
ccaagtcaac cccccggaaa catcatatgg aattcatcag actccaaaat tatcctgaat 2820
tgggatcaag tgaaggccct ggataatgag tcggaagtaa aaggatacaa agtcttgtac 2880
agatggaaca gacaaagcag cacatctgtc attgaaacaa ataaaacatc ggtggagctt 2940
tctttgcctt tcgatgaaga ttatataata gaaattaagc cattcagcga cggaggagat 3000
ggcagcagca gtgaacaaat tcgaattccc gggggaggta gcaaagactg cgaaatgaag 3060
cgcaccaccc tggatagccc tctgggcaag ctggaactgt ctgggtgcga acagggcctg 3120
cacgagatca agctgctggg caaaggaaca tctgccgccg acgccgtgga agtgcctgcc 3180
ccagccgccg tgctgggcgg accagagcca ctgatgcagg ccaccgcctg gctcaacgcc 3240
tactttcacc agcctgaggc catcgaggag ttccctgtgc cagccctgca ccacccagtg 3300
ttccagcagg agagctttac ccgccaggtg ctgtggaaac tgctgaaagt ggtgaagttc 3360
ggagaggtca tcagctacca gcagctggcc gccctggccg gcaatcccgc cgccaccgcc 3420
gccgtgaaaa ccgccctgag cggaaatccc gtgcccattc tgatcccctg ccaccgggtg 3480
gtgtctagct ctggcgccgt ggggggctac gagggcgggc tcgccgtgaa agagtggctg 3540
ctggcccacg agggccacag actgggcaag cctgggctgg gtcctgcagg tataggcgcg 3600
ccagggtccc tggagaatct atattttcaa agtggcggag gtagccatca tcatcatcat 3660
cattgatgac cggtaagctt gcggccgc 3688
<210> 93
<211> 1221
<212> PRT
<213>Artificial sequence
<220>
<223>The amino acid sequence of BiP-CNTN419-990-SNAP-ProTEV-His labels
<400> 93
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Gly Arg Ser Met Arg Leu Pro Trp Glu Leu Leu Val Leu Gln Ser
20 25 30
Phe Ile Leu Cys Leu Ala Asp Asp Ser Thr Leu His Gly Pro Ile Phe
35 40 45
Ile Gln Glu Pro Ser Pro Val Met Phe Pro Leu Asp Ser Glu Glu Lys
50 55 60
Lys Val Lys Leu Asn Cys Glu Val Lys Gly Asn Pro Lys Pro His Ile
65 70 75 80
Arg Trp Lys Leu Asn Gly Thr Asp Val Asp Thr Gly Met Asp Phe Arg
85 90 95
Tyr Ser Val Val Glu Gly Ser Leu Leu Ile Asn Asn Pro Asn Lys Thr
100 105 110
Gln Asp Ala Gly Thr Tyr Gln Cys Thr Ala Thr Asn Ser Phe Gly Thr
115 120 125
Ile Val Ser Arg Glu Ala Lys Leu Gln Phe Ala Tyr Leu Asp Asn Phe
130 135 140
Lys Thr Arg Thr Arg Ser Thr Val Ser Val Arg Arg Gly Gln Gly Met
145 150 155 160
Val Leu Leu Cys Gly Pro Pro Pro His Ser Gly Glu Leu Ser Tyr Ala
165 170 175
Trp Ile Phe Asn Glu Tyr Pro Ser Tyr Gln Asp Asn Arg Arg Phe Val
180 185 190
Ser Gln Glu Thr Gly Asn Leu Tyr Ile Ala Lys Val Glu Lys Ser Asp
195 200 205
Val Gly Asn Tyr Thr Cys Val Val Thr Asn Thr Val Thr Asn His Lys
210 215 220
Val Leu Gly Pro Pro Thr Pro Leu Ile Leu Arg Asn Asp Gly Val Met
225 230 235 240
Gly Glu Tyr Glu Pro Lys Ile Glu Val Gln Phe Pro Glu Thr Val Pro
245 250 255
Thr Ala Lys Gly Ala Thr Val Lys Leu Glu Cys Phe Ala Leu Gly Asn
260 265 270
Pro Val Pro Thr Ile Ile Trp Arg Arg Ala Asp Gly Lys Pro Ile Ala
275 280 285
Arg Lys Ala Arg Arg His Lys Ser Asn Gly Ile Leu Glu Ile Pro Asn
290 295 300
Phe Gln Gln Glu Asp Ala Gly Leu Tyr Glu Cys Val Ala Glu Asn Ser
305 310 315 320
Arg Gly Lys Asn Val Ala Arg Gly Gln Leu Thr Phe Tyr Ala Gln Pro
325 330 335
Asn Trp Ile Gln Lys Ile Asn Asp Ile His Val Ala Met Glu Glu Asn
340 345 350
Val Phe Trp Glu Cys Lys Ala Asn Gly Arg Pro Lys Pro Thr Tyr Lys
355 360 365
Trp Leu Lys Asn Gly Glu Pro Leu Leu Thr Arg Asp Arg Ile Gln Ile
370 375 380
Glu Gln Gly Thr Leu Asn Ile Thr Ile Val Asn Leu Ser Asp Ala Gly
385 390 395 400
Met Tyr Gln Cys Leu Ala Glu Asn Lys His Gly Val Ile Phe Ser Asn
405 410 415
Ala Glu Leu Ser Val Ile Ala Val Gly Pro Asp Phe Ser Arg Thr Leu
420 425 430
Leu Lys Arg Val Thr Leu Val Lys Val Gly Gly Glu Val Val Ile Glu
435 440 445
Cys Lys Pro Lys Ala Ser Pro Lys Pro Val Tyr Thr Trp Lys Lys Gly
450 455 460
Arg Asp Ile Leu Lys Glu Asn Glu Arg Ile Thr Ile Ser Glu Asp Gly
465 470 475 480
Asn Leu Arg Ile Ile Asn Val Thr Lys Ser Asp Ala Gly Ser Tyr Thr
485 490 495
Cys Ile Ala Thr Asn His Phe Gly Thr Ala Ser Ser Thr Gly Asn Leu
500 505 510
Val Val Lys Asp Pro Thr Arg Val Met Val Pro Pro Ser Ser Met Asp
515 520 525
Val Thr Val Gly Glu Ser Ile Val Leu Pro Cys Gln Val Thr His Asp
530 535 540
His Ser Leu Asp Ile Val Phe Thr Trp Ser Phe Asn Gly His Leu Ile
545 550 555 560
Asp Phe Asp Arg Asp Gly Asp His Phe Glu Arg Val Gly Gly Gln Asp
565 570 575
Ser Ala Gly Asp Leu Met Ile Arg Asn Ile Gln Leu Lys His Ala Gly
580 585 590
Lys Tyr Val Cys Met Val Gln Thr Ser Val Asp Arg Leu Ser Ala Ala
595 600 605
Ala Asp Leu Ile Val Arg Gly Pro Pro Gly Pro Pro Glu Ala Val Thr
610 615 620
Ile Asp Glu Ile Thr Asp Thr Thr Ala Gln Leu Ser Trp Arg Pro Gly
625 630 635 640
Pro Asp Asn His Ser Pro Ile Thr Met Tyr Val Ile Gln Ala Arg Thr
645 650 655
Pro Phe Ser Val Gly Trp Gln Ala Val Ser Thr Val Pro Glu Leu Ile
660 665 670
Asp Gly Lys Thr Phe Thr Ala Thr Val Val Gly Leu Asn Pro Trp Val
675 680 685
Glu Tyr Glu Phe Arg Thr Val Ala Ala Asn Val Ile Gly Ile Gly Glu
690 695 700
Pro Ser Arg Pro Ser Glu Lys Arg Arg Thr Glu Glu Ala Leu Pro Glu
705 710 715 720
Val Thr Pro Ala Asn Val Ser Gly Gly Gly Gly Ser Lys Ser Glu Leu
725 730 735
Val Ile Thr Trp Glu Thr Val Pro Glu Glu Leu Gln Asn Gly Arg Gly
740 745 750
Phe Gly Tyr Val Val Ala Phe Arg Pro Tyr Gly Lys Met Ile Trp Met
755 760 765
Leu Thr Val Leu Ala Ser Ala Asp Ala Ser Arg Tyr Val Phe Arg Asn
770 775 780
Glu Ser Val His Pro Phe Ser Pro Phe Glu Val Lys Val Gly Val Phe
785 790 795 800
Asn Asn Lys Gly Glu Gly Pro Phe Ser Pro Thr Thr Val Val Tyr Ser
805 810 815
Ala Glu Glu Glu Pro Thr Lys Pro Pro Ala Ser Ile Phe Ala Arg Ser
820 825 830
Leu Ser Ala Thr Asp Ile Glu Val Phe Trp Ala Ser Pro Leu Glu Lys
835 840 845
Asn Arg Gly Arg Ile Gln Gly Tyr Glu Val Lys Tyr Trp Arg His Glu
850 855 860
Asp Lys Glu Glu Asn Ala Arg Lys Ile Arg Thr Val Gly Asn Gln Thr
865 870 875 880
Ser Thr Lys Ile Thr Asn Leu Lys Gly Ser Val Leu Tyr His Leu Ala
885 890 895
Val Lys Ala Tyr Asn Ser Ala Gly Thr Gly Pro Ser Ser Ala Thr Val
900 905 910
Asn Val Thr Thr Arg Lys Pro Pro Pro Ser Gln Pro Pro Gly Asn Ile
915 920 925
Ile Trp Asn Ser Ser Asp Ser Lys Ile Ile Leu Asn Trp Asp Gln Val
930 935 940
Lys Ala Leu Asp Asn Glu Ser Glu Val Lys Gly Tyr Lys Val Leu Tyr
945 950 955 960
Arg Trp Asn Arg Gln Ser Ser Thr Ser Val Ile Glu Thr Asn Lys Thr
965 970 975
Ser Val Glu Leu Ser Leu Pro Phe Asp Glu Asp Tyr Ile Ile Glu Ile
980 985 990
Lys Pro Phe Ser Asp Gly Gly Asp Gly Ser Ser Ser Glu Gln Ile Arg
995 1000 1005
Ile Pro Gly Gly Gly Ser Lys Asp Cys Glu Met Lys Arg Thr Thr
1010 1015 1020
Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln
1025 1030 1035
Gly Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala
1040 1045 1050
Asp Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro
1055 1060 1065
Glu Pro Leu Met Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His
1070 1075 1080
Gln Pro Glu Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His
1085 1090 1095
Pro Val Phe Gln Gln Glu Ser Phe Thr Arg Gln Val Leu Trp Lys
1100 1105 1110
Leu Leu Lys Val Val Lys Phe Gly Glu Val Ile Ser Tyr Gln Gln
1115 1120 1125
Leu Ala Ala Leu Ala Gly Asn Pro Ala Ala Thr Ala Ala Val Lys
1130 1135 1140
Thr Ala Leu Ser Gly Asn Pro Val Pro Ile Leu Ile Pro Cys His
1145 1150 1155
Arg Val Val Ser Ser Ser Gly Ala Val Gly Gly Tyr Glu Gly Gly
1160 1165 1170
Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu Gly His Arg Leu
1175 1180 1185
Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala Pro Gly Ser
1190 1195 1200
Leu Glu Asn Leu Tyr Phe Gln Ser Gly Gly Gly Ser His His His
1205 1210 1215
His His His
1220
<210> 94
<211> 2538
<212> DNA
<213>People
<400> 94
ttcctgtcgc accaccgcct gaaaggcagg tttcagaggg accgcaggaa catccgcccc 60
aacatcatcc tggtgctgac ggacgaccag gatgtggagc tgggttccat gcaggtgatg 120
aacaagaccc ggcgcatcat ggagcagggc ggggcgcact tcatcaacgc cttcgtgacc 180
acacccatgt gctgcccctc acgctcctcc atcctcactg gcaagtacgt ccacaaccac 240
aacacctaca ccaacaatga gaactgctcc tcgccctcct ggcaggcaca gcacgagagc 300
cgcacctttg ccgtgtacct caatagcact ggctaccgga cagctttctt cgggaagtat 360
cttaatgaat acaacggctc ctacgtgcca cccggctgga aggagtgggt cggactcctt 420
aaaaactccc gcttttataa ctacacgctg tgtcggaacg gggtgaaaga gaagcacggc 480
tccgactact ccaaggatta cctcacagac ctcatcacca atgacagcgt gagcttcttc 540
cgcacgtcca agaagatgta cccgcacagg ccagtcctca tggtcatcag ccatgcagcc 600
ccccacggcc ctgaggattc agccccacaa tattcacgcc tcttcccaaa cgcatctcag 660
cacatcacgc cgagctacaa ctacgcgccc aacccggaca aacactggat catgcgctac 720
acggggccca tgaagcccat ccacatggaa ttcaccaaca tgctccagcg gaagcgcttg 780
cagaccctca tgtcggtgga cgactccatg gagacgattt acaacatgct ggttgagacg 840
ggcgagctgg acaacacgta catcgtatac accgccgacc acggttacca catcggccag 900
tttggcctgg tgaaagggaa atccatgcca tatgagtttg acatcagggt cccgttctac 960
gtgaggggcc ccaacgtgga agccggctgt ctgaatcccc acatcgtcct caacattgac 1020
ctggccccca ccatcctgga cattgcaggc ctggacatac ctgcggatat ggacgggaaa 1080
tccatcctca agctgctgga cacggagcgg ccggtgaatc ggtttcactt gaaaaagaag 1140
atgagggtct ggcgggactc cttcttggtg gagagaggca agctgctaca caagagagac 1200
aatgacaagg tggacgccca ggaggagaac tttctgccca agtaccagcg tgtgaaggac 1260
ctgtgtcagc gtgctgagta ccagacggcg tgtgagcagc tgggacagaa gtggcagtgt 1320
gtggaggacg ccacggggaa gctgaagctg cataagtgca agggccccat gcggctgggc 1380
ggcagcagag ccctctccaa cctcgtgccc aagtactacg ggcagggcag cgaggcctgc 1440
acctgtgaca gcggggacta caagctcagc ctggccggac gccggaaaaa actcttcaag 1500
aagaagtaca aggccagcta tgtccgcagt cgctccatcc gctcagtggc catcgaggtg 1560
gacggcaggg tgtaccacgt aggcctgggt gatgccgccc agccccgaaa cctcaccaag 1620
cggcactggc caggggcccc tgaggaccaa gatgacaagg atggtgggga cttcagtggc 1680
actggaggcc ttcccgacta ctcagccgcc aaccccatta aagtgacaca tcggtgctac 1740
atcctagaga acgacacagt ccagtgtgac ctggacctgt acaagtccct gcaggcctgg 1800
aaagaccaca agctgcacat cgaccacgag attgaaaccc tgcagaacaa aattaagaac 1860
ctgagggaag tccgaggtca cctgaagaaa aagcggccag aagaatgtga ctgtcacaaa 1920
atcagctacc acacccagca caaaggccgc ctcaagcaca gaggctccag tctgcatcct 1980
ttcaggaagg gcctgcaaga gaaggacaag gtgtggctgt tgcgggagca gaagcgcaag 2040
aagaaactcc gcaagctgct caagcgcctg cagaacaacg acacgtgcag catgccaggc 2100
ctcacgtgct tcacccacga caaccagcac tggcagacgg cgcctttctg gacactgggg 2160
cctttctgtg cctgcaccag cgccaacaat aacacgtact ggtgcatgag gaccatcaat 2220
gagactcaca atttcctctt ctgtgaattt gcaactggct tcctagagta ctttgatctc 2280
aacacagacc cctaccagct gatgaatgca gtgaacacac tggacaggga tgtcctcaac 2340
cagctacacg tacagctcat ggagctgagg agctgcaagg gttacaagca gtgtaacccc 2400
cggactcgaa acatggacct gggacttaaa gatggaggaa gctatgagca atacaggcag 2460
tttcagcgtc gaaagtggcc agaaatgaag agaccttctt ccaaatcact gggacaactg 2520
tgggaaggct gggaaggc 2538
<210> 95
<211> 879
<212> PRT
<213>People
<400> 95
Gly His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly Ile Gly Ala
1 5 10 15
Pro Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Ser Asp
20 25 30
Ile Phe Leu Ser His His Arg Leu Lys Gly Arg Phe Gln Arg Asp Arg
35 40 45
Arg Asn Ile Arg Pro Asn Ile Ile Leu Val Leu Thr Asp Asp Gln Asp
50 55 60
Val Glu Leu Gly Ser Met Gln Val Met Asn Lys Thr Arg Arg Ile Met
65 70 75 80
Glu Gln Gly Gly Ala His Phe Ile Asn Ala Phe Val Thr Thr Pro Met
85 90 95
Cys Cys Pro Ser Arg Ser Ser Ile Leu Thr Gly Lys Tyr Val His Asn
100 105 110
His Asn Thr Tyr Thr Asn Asn Glu Asn Cys Ser Ser Pro Ser Trp Gln
115 120 125
Ala Gln His Glu Ser Arg Thr Phe Ala Val Tyr Leu Asn Ser Thr Gly
130 135 140
Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu Asn Glu Tyr Asn Gly Ser
145 150 155 160
Tyr Val Pro Pro Gly Trp Lys Glu Trp Val Gly Leu Leu Lys Asn Ser
165 170 175
Arg Phe Tyr Asn Tyr Thr Leu Cys Arg Asn Gly Val Lys Glu Lys His
180 185 190
Gly Ser Asp Tyr Ser Lys Asp Tyr Leu Thr Asp Leu Ile Thr Asn Asp
195 200 205
Ser Val Ser Phe Phe Arg Thr Ser Lys Lys Met Tyr Pro His Arg Pro
210 215 220
Val Leu Met Val Ile Ser His Ala Ala Pro His Gly Pro Glu Asp Ser
225 230 235 240
Ala Pro Gln Tyr Ser Arg Leu Phe Pro Asn Ala Ser Gln His Ile Thr
245 250 255
Pro Ser Tyr Asn Tyr Ala Pro Asn Pro Asp Lys His Trp Ile Met Arg
260 265 270
Tyr Thr Gly Pro Met Lys Pro Ile His Met Glu Phe Thr Asn Met Leu
275 280 285
Gln Arg Lys Arg Leu Gln Thr Leu Met Ser Val Asp Asp Ser Met Glu
290 295 300
Thr Ile Tyr Asn Met Leu Val Glu Thr Gly Glu Leu Asp Asn Thr Tyr
305 310 315 320
Ile Val Tyr Thr Ala Asp His Gly Tyr His Ile Gly Gln Phe Gly Leu
325 330 335
Val Lys Gly Lys Ser Met Pro Tyr Glu Phe Asp Ile Arg Val Pro Phe
340 345 350
Tyr Val Arg Gly Pro Asn Val Glu Ala Gly Cys Leu Asn Pro His Ile
355 360 365
Val Leu Asn Ile Asp Leu Ala Pro Thr Ile Leu Asp Ile Ala Gly Leu
370 375 380
Asp Ile Pro Ala Asp Met Asp Gly Lys Ser Ile Leu Lys Leu Leu Asp
385 390 395 400
Thr Glu Arg Pro Val Asn Arg Phe His Leu Lys Lys Lys Met Arg Val
405 410 415
Trp Arg Asp Ser Phe Leu Val Glu Arg Gly Lys Leu Leu His Lys Arg
420 425 430
Asp Asn Asp Lys Val Asp Ala Gln Glu Glu Asn Phe Leu Pro Lys Tyr
435 440 445
Gln Arg Val Lys Asp Leu Cys Gln Arg Ala Glu Tyr Gln Thr Ala Cys
450 455 460
Glu Gln Leu Gly Gln Lys Trp Gln Cys Val Glu Asp Ala Thr Gly Lys
465 470 475 480
Leu Lys Leu His Lys Cys Lys Gly Pro Met Arg Leu Gly Gly Ser Arg
485 490 495
Ala Leu Ser Asn Leu Val Pro Lys Tyr Tyr Gly Gln Gly Ser Glu Ala
500 505 510
Cys Thr Cys Asp Ser Gly Asp Tyr Lys Leu Ser Leu Ala Gly Arg Arg
515 520 525
Lys Lys Leu Phe Lys Lys Lys Tyr Lys Ala Ser Tyr Val Arg Ser Arg
530 535 540
Ser Ile Arg Ser Val Ala Ile Glu Val Asp Gly Arg Val Tyr His Val
545 550 555 560
Gly Leu Gly Asp Ala Ala Gln Pro Arg Asn Leu Thr Lys Arg His Trp
565 570 575
Pro Gly Ala Pro Glu Asp Gln Asp Asp Lys Asp Gly Gly Asp Phe Ser
580 585 590
Gly Thr Gly Gly Leu Pro Asp Tyr Ser Ala Ala Asn Pro Ile Lys Val
595 600 605
Thr His Arg Cys Tyr Ile Leu Glu Asn Asp Thr Val Gln Cys Asp Leu
610 615 620
Asp Leu Tyr Lys Ser Leu Gln Ala Trp Lys Asp His Lys Leu His Ile
625 630 635 640
Asp His Glu Ile Glu Thr Leu Gln Asn Lys Ile Lys Asn Leu Arg Glu
645 650 655
Val Arg Gly His Leu Lys Lys Lys Arg Pro Glu Glu Cys Asp Cys His
660 665 670
Lys Ile Ser Tyr His Thr Gln His Lys Gly Arg Leu Lys His Arg Gly
675 680 685
Ser Ser Leu His Pro Phe Arg Lys Gly Leu Gln Glu Lys Asp Lys Val
690 695 700
Trp Leu Leu Arg Glu Gln Lys Arg Lys Lys Lys Leu Arg Lys Leu Leu
705 710 715 720
Lys Arg Leu Gln Asn Asn Asp Thr Cys Ser Met Pro Gly Leu Thr Cys
725 730 735
Phe Thr His Asp Asn Gln His Trp Gln Thr Ala Pro Phe Trp Thr Leu
740 745 750
Gly Pro Phe Cys Ala Cys Thr Ser Ala Asn Asn Asn Thr Tyr Trp Cys
755 760 765
Met Arg Thr Ile Asn Glu Thr His Asn Phe Leu Phe Cys Glu Phe Ala
770 775 780
Thr Gly Phe Leu Glu Tyr Phe Asp Leu Asn Thr Asp Pro Tyr Gln Leu
785 790 795 800
Met Asn Ala Val Asn Thr Leu Asp Arg Asp Val Leu Asn Gln Leu His
805 810 815
Val Gln Leu Met Glu Leu Arg Ser Cys Lys Gly Tyr Lys Gln Cys Asn
820 825 830
Pro Arg Thr Arg Asn Met Asp Leu Gly Leu Lys Asp Gly Gly Ser Tyr
835 840 845
Glu Gln Tyr Arg Gln Phe Gln Arg Arg Lys Trp Pro Glu Met Lys Arg
850 855 860
Pro Ser Ser Lys Ser Leu Gly Gln Leu Trp Glu Gly Trp Glu Gly
865 870 875
<210> 96
<211> 3322
<212> DNA
<213>Artificial sequence
<220>
<223>BiP samples SNAP-hSULF2 DNA sequence dna
<400> 96
tcgcgagcta gcaccatgaa actatgtatt ctacttgcag ttgttgcgtt cgtaggattg 60
tccttaccta cagctctggc aagatctgac aaagactgcg aaatgaaaag aactacattg 120
gattcaccac ttgggaagtt ggaactgagt ggatgcgagc aaggattgca tgaaattaag 180
ctactgggaa aaggaacttc tgctgctgat gcagttgaag ttccagcacc agcagctgtt 240
cttggaggtc ctgagcccct catgcaagcc acagcctggc ttaacgcata tttccaccag 300
cctgaggcca ttgaggaatt tccagtcccc gcccttcacc atcctgtgtt tcagcaggag 360
agcttcaccc gccaggtcct gtggaaattg ctgaaggtgg tcaagtttgg tgaagtgatt 420
tcatatcagc aacttgctgc attggccggt aaccccgcag ctacagctgc cgtgaaaact 480
gctctcagcg gaaatcctgt gcccatcctg atcccttgtc acagagtcgt ttcatcttcc 540
ggagctgtag gtggctatga aggaggactg gcagttaagg agtggctgct ggctcatgaa 600
ggtcatagac ttggaaagcc tgggctgggt cctgctggta taggcgcgcc agggtcccta 660
ggtggcggat ccgaaaacct gtacttccag agcgatatct tcctgtcgca ccaccgcctg 720
aaaggcaggt ttcagaggga ccgcaggaac atccgcccca acatcatcct ggtgctgacg 780
gacgaccagg atgtggagct gggttccatg caggtgatga acaagacccg gcgcatcatg 840
gagcagggcg gggcgcactt catcaacgcc ttcgtgacca cacccatgtg ctgcccctca 900
cgctcctcca tcctcactgg caagtacgtc cacaaccaca acacctacac caacaatgag 960
aactgctcct cgccctcctg gcaggcacag cacgagagcc gcacctttgc cgtgtacctc 1020
aatagcactg gctaccggac agctttcttc gggaagtatc ttaatgaata caacggctcc 1080
tacgtgccac ccggctggaa ggagtgggtc ggactcctta aaaactcccg cttttataac 1140
tacacgctgt gtcggaacgg ggtgaaagag aagcacggct ccgactactc caaggattac 1200
ctcacagacc tcatcaccaa tgacagcgtg agcttcttcc gcacgtccaa gaagatgtac 1260
ccgcacaggc cagtcctcat ggtcatcagc catgcagccc cccacggccc tgaggattca 1320
gccccacaat attcacgcct cttcccaaac gcatctcagc acatcacgcc gagctacaac 1380
tacgcgccca acccggacaa acactggatc atgcgctaca cggggcccat gaagcccatc 1440
cacatggaat tcaccaacat gctccagcgg aagcgcttgc agaccctcat gtcggtggac 1500
gactccatgg agacgattta caacatgctg gttgagacgg gcgagctgga caacacgtac 1560
atcgtataca ccgccgacca cggttaccac atcggccagt ttggcctggt gaaagggaaa 1620
tccatgccat atgagtttga catcagggtc ccgttctacg tgaggggccc caacgtggaa 1680
gccggctgtc tgaatcccca catcgtcctc aacattgacc tggcccccac catcctggac 1740
attgcaggcc tggacatacc tgcggatatg gacgggaaat ccatcctcaa gctgctggac 1800
acggagcggc cggtgaatcg gtttcacttg aaaaagaaga tgagggtctg gcgggactcc 1860
ttcttggtgg agagaggcaa gctgctacac aagagagaca atgacaaggt ggacgcccag 1920
gaggagaact ttctgcccaa gtaccagcgt gtgaaggacc tgtgtcagcg tgctgagtac 1980
cagacggcgt gtgagcagct gggacagaag tggcagtgtg tggaggacgc cacggggaag 2040
ctgaagctgc ataagtgcaa gggccccatg cggctgggcg gcagcagagc cctctccaac 2100
ctcgtgccca agtactacgg gcagggcagc gaggcctgca cctgtgacag cggggactac 2160
aagctcagcc tggccggacg ccggaaaaaa ctcttcaaga agaagtacaa ggccagctat 2220
gtccgcagtc gctccatccg ctcagtggcc atcgaggtgg acggcagggt gtaccacgta 2280
ggcctgggtg atgccgccca gccccgaaac ctcaccaagc ggcactggcc aggggcccct 2340
gaggaccaag atgacaagga tggtggggac ttcagtggca ctggaggcct tcccgactac 2400
tcagccgcca accccattaa agtgacacat cggtgctaca tcctagagaa cgacacagtc 2460
cagtgtgacc tggacctgta caagtccctg caggcctgga aagaccacaa gctgcacatc 2520
gaccacgaga ttgaaaccct gcagaacaaa attaagaacc tgagggaagt ccgaggtcac 2580
ctgaagaaaa agcggccaga agaatgtgac tgtcacaaaa tcagctacca cacccagcac 2640
aaaggccgcc tcaagcacag aggctccagt ctgcatcctt tcaggaaggg cctgcaagag 2700
aaggacaagg tgtggctgtt gcgggagcag aagcgcaaga agaaactccg caagctgctc 2760
aagcgcctgc agaacaacga cacgtgcagc atgccaggcc tcacgtgctt cacccacgac 2820
aaccagcact ggcagacggc gcctttctgg acactggggc ctttctgtgc ctgcaccagc 2880
gccaacaata acacgtactg gtgcatgagg accatcaatg agactcacaa tttcctcttc 2940
tgtgaatttg caactggctt cctagagtac tttgatctca acacagaccc ctaccagctg 3000
atgaatgcag tgaacacact ggacagggat gtcctcaacc agctacacgt acagctcatg 3060
gagctgagga gctgcaaggg ttacaagcag tgtaaccccc ggactcgaaa catggacctg 3120
ggacttaaag atggaggaag ctatgagcaa tacaggcagt ttcagcgtcg aaagtggcca 3180
gaaatgaaga gaccttcttc caaatcactg ggacaactgt gggaaggctg ggaaggcccg 3240
ggagagaatc tatattttca agggcccggc ggaggtagtc accatcatca ccatcactaa 3300
tgaccggtgc ggccgcaagc tt 3322
<210> 97
<211> 1094
<212> PRT
<213>Artificial sequence
<220>
<223>BiP samples SNAP-hSULF2 amino acid sequence
<400> 97
Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser
1 5 10 15
Leu Pro Thr Ala Leu Ala Arg Ser Asp Lys Asp Cys Glu Met Lys Arg
20 25 30
Thr Thr Leu Asp Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu
35 40 45
Gln Gly Leu His Glu Ile Lys Leu Leu Gly Lys Gly Thr Ser Ala Ala
50 55 60
Asp Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu
65 70 75 80
Pro Leu Met Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro
85 90 95
Glu Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe
100 105 110
Gln Gln Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val
115 120 125
Val Lys Phe Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu Ala
130 135 140
Gly Asn Pro Ala Ala Thr Ala Ala Val Lys Thr Ala Leu Ser Gly Asn
145 150 155 160
Pro Val Pro Ile Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly
165 170 175
Ala Val Gly Gly Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu
180 185 190
Ala His Glu Gly His Arg Leu Gly Lys Pro Gly Leu Gly Pro Ala Gly
195 200 205
Ile Gly Ala Pro Gly Ser Leu Gly Gly Gly Ser Glu Asn Leu Tyr Phe
210 215 220
Gln Ser Asp Ile Phe Leu Ser His His Arg Leu Lys Gly Arg Phe Gln
225 230 235 240
Arg Asp Arg Arg Asn Ile Arg Pro Asn Ile Ile Leu Val Leu Thr Asp
245 250 255
Asp Gln Asp Val Glu Leu Gly Ser Met Gln Val Met Asn Lys Thr Arg
260 265 270
Arg Ile Met Glu Gln Gly Gly Ala His Phe Ile Asn Ala Phe Val Thr
275 280 285
Thr Pro Met Cys Cys Pro Ser Arg Ser Ser Ile Leu Thr Gly Lys Tyr
290 295 300
Val His Asn His Asn Thr Tyr Thr Asn Asn Glu Asn Cys Ser Ser Pro
305 310 315 320
Ser Trp Gln Ala Gln His Glu Ser Arg Thr Phe Ala Val Tyr Leu Asn
325 330 335
Ser Thr Gly Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu Asn Glu Tyr
340 345 350
Asn Gly Ser Tyr Val Pro Pro Gly Trp Lys Glu Trp Val Gly Leu Leu
355 360 365
Lys Asn Ser Arg Phe Tyr Asn Tyr Thr Leu Cys Arg Asn Gly Val Lys
370 375 380
Glu Lys His Gly Ser Asp Tyr Ser Lys Asp Tyr Leu Thr Asp Leu Ile
385 390 395 400
Thr Asn Asp Ser Val Ser Phe Phe Arg Thr Ser Lys Lys Met Tyr Pro
405 410 415
His Arg Pro Val Leu Met Val Ile Ser His Ala Ala Pro His Gly Pro
420 425 430
Glu Asp Ser Ala Pro Gln Tyr Ser Arg Leu Phe Pro Asn Ala Ser Gln
435 440 445
His Ile Thr Pro Ser Tyr Asn Tyr Ala Pro Asn Pro Asp Lys His Trp
450 455 460
Ile Met Arg Tyr Thr Gly Pro Met Lys Pro Ile His Met Glu Phe Thr
465 470 475 480
Asn Met Leu Gln Arg Lys Arg Leu Gln Thr Leu Met Ser Val Asp Asp
485 490 495
Ser Met Glu Thr Ile Tyr Asn Met Leu Val Glu Thr Gly Glu Leu Asp
500 505 510
Asn Thr Tyr Ile Val Tyr Thr Ala Asp His Gly Tyr His Ile Gly Gln
515 520 525
Phe Gly Leu Val Lys Gly Lys Ser Met Pro Tyr Glu Phe Asp Ile Arg
530 535 540
Val Pro Phe Tyr Val Arg Gly Pro Asn Val Glu Ala Gly Cys Leu Asn
545 550 555 560
Pro His Ile Val Leu Asn Ile Asp Leu Ala Pro Thr Ile Leu Asp Ile
565 570 575
Ala Gly Leu Asp Ile Pro Ala Asp Met Asp Gly Lys Ser Ile Leu Lys
580 585 590
Leu Leu Asp Thr Glu Arg Pro Val Asn Arg Phe His Leu Lys Lys Lys
595 600 605
Met Arg Val Trp Arg Asp Ser Phe Leu Val Glu Arg Gly Lys Leu Leu
610 615 620
His Lys Arg Asp Asn Asp Lys Val Asp Ala Gln Glu Glu Asn Phe Leu
625 630 635 640
Pro Lys Tyr Gln Arg Val Lys Asp Leu Cys Gln Arg Ala Glu Tyr Gln
645 650 655
Thr Ala Cys Glu Gln Leu Gly Gln Lys Trp Gln Cys Val Glu Asp Ala
660 665 670
Thr Gly Lys Leu Lys Leu His Lys Cys Lys Gly Pro Met Arg Leu Gly
675 680 685
Gly Ser Arg Ala Leu Ser Asn Leu Val Pro Lys Tyr Tyr Gly Gln Gly
690 695 700
Ser Glu Ala Cys Thr Cys Asp Ser Gly Asp Tyr Lys Leu Ser Leu Ala
705 710 715 720
Gly Arg Arg Lys Lys Leu Phe Lys Lys Lys Tyr Lys Ala Ser Tyr Val
725 730 735
Arg Ser Arg Ser Ile Arg Ser Val Ala Ile Glu Val Asp Gly Arg Val
740 745 750
Tyr His Val Gly Leu Gly Asp Ala Ala Gln Pro Arg Asn Leu Thr Lys
755 760 765
Arg His Trp Pro Gly Ala Pro Glu Asp Gln Asp Asp Lys Asp Gly Gly
770 775 780
Asp Phe Ser Gly Thr Gly Gly Leu Pro Asp Tyr Ser Ala Ala Asn Pro
785 790 795 800
Ile Lys Val Thr His Arg Cys Tyr Ile Leu Glu Asn Asp Thr Val Gln
805 810 815
Cys Asp Leu Asp Leu Tyr Lys Ser Leu Gln Ala Trp Lys Asp His Lys
820 825 830
Leu His Ile Asp His Glu Ile Glu Thr Leu Gln Asn Lys Ile Lys Asn
835 840 845
Leu Arg Glu Val Arg Gly His Leu Lys Lys Lys Arg Pro Glu Glu Cys
850 855 860
Asp Cys His Lys Ile Ser Tyr His Thr Gln His Lys Gly Arg Leu Lys
865 870 875 880
His Arg Gly Ser Ser Leu His Pro Phe Arg Lys Gly Leu Gln Glu Lys
885 890 895
Asp Lys Val Trp Leu Leu Arg Glu Gln Lys Arg Lys Lys Lys Leu Arg
900 905 910
Lys Leu Leu Lys Arg Leu Gln Asn Asn Asp Thr Cys Ser Met Pro Gly
915 920 925
Leu Thr Cys Phe Thr His Asp Asn Gln His Trp Gln Thr Ala Pro Phe
930 935 940
Trp Thr Leu Gly Pro Phe Cys Ala Cys Thr Ser Ala Asn Asn Asn Thr
945 950 955 960
Tyr Trp Cys Met Arg Thr Ile Asn Glu Thr His Asn Phe Leu Phe Cys
965 970 975
Glu Phe Ala Thr Gly Phe Leu Glu Tyr Phe Asp Leu Asn Thr Asp Pro
980 985 990
Tyr Gln Leu Met Asn Ala Val Asn Thr Leu Asp Arg Asp Val Leu Asn
995 1000 1005
Gln Leu His Val Gln Leu Met Glu Leu Arg Ser Cys Lys Gly Tyr
1010 1015 1020
Lys Gln Cys Asn Pro Arg Thr Arg Asn Met Asp Leu Gly Leu Lys
1025 1030 1035
Asp Gly Gly Ser Tyr Glu Gln Tyr Arg Gln Phe Gln Arg Arg Lys
1040 1045 1050
Trp Pro Glu Met Lys Arg Pro Ser Ser Lys Ser Leu Gly Gln Leu
1055 1060 1065
Trp Glu Gly Trp Glu Gly Pro Gly Glu Asn Leu Tyr Phe Gln Gly
1070 1075 1080
Pro Gly Gly Gly Ser His His His His His His
1085 1090
Claims (38)
1.SEQ ID NO：6- methyl guanine-DNA- methyltransferase mutants shown in 2 are used to strengthen heterologous protein in sense
The purposes for the production being infected with the host cell of science or defective vector.
2. purposes according to claim 1, wherein the heterologous protein is with least 40mg/ of the cells and supernatant reclaimed
L or more is expressed.
3. purposes according to claim 1, wherein the heterologous protein is expressed in the form of fused polypeptide, the fusion is more
Peptide is comprised at least：
I) have functional peptide secretion signal in the host cell,
Ii) 6- methyl guanines-DNA- the methyltransferase mutants, and
Iii) the heterologous protein.
4. purposes according to claim 1, wherein the heterologous protein is as coded by carrier, the carrier is included in single
The following nucleotide sequence encoded in ORFs, according to 5' to 3' direction：
A) have functional peptide secretion signal in the host cell,
B) the 6- methyl guanines-DNA- methyltransferase mutants, and
C) heterologous protein.
5. purposes according to claim 4, wherein the ORFs is operationally combined with inducible promoter, institute
It is functional to state inducible promoter same with peptide signal in identical host cell.
6. purposes according to claim 5, wherein the secretion peptide signal and the inducible promoter are thin in drosophila S2
It is functional in born of the same parents.
7. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is bacterial immunogenic albumen or disease
Malicious immunogenic protein.
8. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is cell factor.
9. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is anti-tumor protein.
10. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is antigen.
11. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is antimicrobial polypeptide.
12. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is virus polypeptide.
13. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is parasite polypeptide.
14. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is anticoagulant.
15. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is hormone.
16. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is therapeutic enzyme.
17. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is monoclonal antibody.
18. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is blood factor.
19. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is growth factor.
20. purposes according to claim 7, wherein the bacterial immunogenic albumen or the choosing of viral immunogenic albumen
From：
Encephalitis, yellow heat, Usu figure, sieve Theo, Murray encephalitis, Wei Saiersi Blang, hereby are propagated from Dengue, encephalitis B, tick
Card or the EDIII albumen of west nile virus,
Nucleoprotein N from Rift Valley fever or toscana virus,
The soluble form of E2 envelope proteins from chikungunya virus,
The soluble form of west nile virus E envelope proteins.
21. purposes according to claim 10, wherein the antigen is cancer antigen.
22. the purposes according to any one of claim 1 to 6, wherein the heterologous protein is selected from：IFN-α, granzyme M,
FasL、SSX2、NERCMSL、hSULF2ΔTMDAnd CNTN4.
23. purposes according to claim 1, wherein the 6- methyl guanines-DNA- methyltransferase mutants are by SEQ
ID NO：1 or SEQ ID NO：47 DNA sequence encoding.
24. purposes according to claim 4, wherein the carrier also encodes at least one peptide cleavage site.
25. purposes according to claim 24, wherein at least one described peptide cleavage site be located at 6- methyl guanines-
Between DNA- methyltransferase mutants and heterologous protein.
26. purposes according to claim 4, wherein the carrier also coded markings.
27. purposes according to claim 26, wherein the marker bit is in the C-terminal of heterologous protein.
28. purposes according to claim 26, wherein the carrier also encoded interval sequence.
29. purposes according to claim 28, wherein the intervening sequence is located at the 6- methyl guanines-DNA- methyl
Shift between enzyme mutant and the heterologous protein, and/or between the heterologous protein and the mark.
30. purposes according to claim 1, wherein the heterologous protein is as coded by carrier, the carrier is included in single
The following nucleotide sequence encoded in one ORFs, according to 5' to 3' direction：
- peptide BiP insect signals,
-SEQ ID NO：SNAP albumen shown in 2,
The heterologous protein defined in-claim 22,
- enterokinase peptide cleavage site,
- polyhistidine is marked, and
- two intervening sequences with amino acid sequence Gly-Gly-Gly-serine.
31. purposes according to claim 1, wherein the heterologous protein is as coded by carrier, the carrier is included in single
The following nucleotide sequence encoded in one ORFs, according to 5' to 3' direction：
- BiP sample peptide signals,
-SEQ ID NO：SNAP albumen shown in 2,
The heterologous protein defined in-claim 22,
- proTEV peptide cleavage sites,
- polyhistidine is marked, and
- two intervening sequences with amino acid sequence Gly-Gly-Gly-serine.
32. purposes according to claim 1, wherein the heterologous protein is as coded by carrier, the carrier is included in single
The following nucleotide sequence encoded in one ORFs, according to 5' to 3' direction：
A) BiP samples peptide signal,
b)SEQ ID NO：SNAP albumen shown in 2,
C) two proTEV peptide cleavage sites,
D) polyhistidine is marked, and
E) two intervening sequences with amino acid sequence Gly-Gly-Gly-serine.
33. purposes according to claim 4, wherein the carrier is expressed in recombinant cell.
34. purposes according to claim 33, wherein the carrier includes SEQ ID NO：59 or SEQ ID NO：69.
35. purposes according to claim 33, wherein the recombinant cell is insect cell.
36. purposes according to claim 35, wherein the insect cell is Drosophila S 2 cells.
37. purposes according to claim 33, wherein the recombinant cell is mammalian cell.
38. purposes according to claim 36, it is characterised in that：
I) expression vector is selected from：
- include SEQ ID NO：19 carrier or the nucleotide sequence being cloned into cell, cell in August in 2010 19 days with
Numbering CNCM I-4357 are deposited in French national Organism Depositary, and Institute Pasteur, Paris is French,
- include SEQ ID NO：22 carrier or the nucleotide sequence being cloned into cell, cell is on October 27th, 2010
French national Organism Depositary is deposited in numbering CNCM I-4381, Institute Pasteur,
- include SEQ ID NO：21 carrier or the nucleotide sequence being cloned into cell, cell is on October 27th, 2010
French national Organism Depositary is deposited in numbering CNCM I-4382, Institute Pasteur,
- include SEQ ID NO：9 carrier or the nucleotide sequence being cloned into cell, cell in September in 2010 29 days with
Numbering CNCM I-4368 are deposited in French national Organism Depositary, Institute Pasteur, and
- include SEQ ID NO：20 carrier or the nucleotide sequence being cloned into cell, cell in September in 2010 29 days with
Numbering CNCM I-4369 are deposited in French national Organism Depositary, Institute Pasteur,
- include SEQ ID NO：The carrier that 10 or 59 or 69 claim 24 is limited,
-SEQ ID NO：64 carrier or the nucleotide sequence being cloned into cell, cell is on December 9th, 2011 with numbering
CNCM I-4581 are deposited in French national Organism Depositary, Institute Pasteur,
-SEQ ID NO：71 carrier,
- include SEQ ID NO：55、SEQ ID NO：57 or 72 or 74, SEQ ID NO：77th, 79 or 81, SEQ ID NO：89、
SEQ ID NO：84 or 86, SEQ ID NO：92 or SEQ ID NO：The carrier that 96 claim 13 is limited,
Or
Ii) it is selected from：
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4357 in August in 2010 19 days
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4381 on October 27th, 2010
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4382 on October 27th, 2010
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4368 in September in 2010 29 days
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4369 in September in 2010 29 days
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4565 on December 5th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4566 on December 5th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4567 on December 5th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4568 on December 5th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4569 on December 5th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4570 on December 5th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4571 on December 5th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4572 on December 5th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4576 on December 8th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4577 on December 8th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4578 on December 8th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4579 on December 8th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4580 on December 8th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4583 on December 9th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4584 on December 9th, 2011
Cell,
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4585 on December 9th, 2011
Cell, and
- French national Organism Depositary, Pasteur research be deposited in numbering CNCM I-4586 on December 9th, 2011
Cell.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP10306389 | 2010-12-09 | ||
| EP10306389.7 | 2010-12-09 | ||
| US201161505694P | 2011-07-08 | 2011-07-08 | |
| US61/505,694 | 2011-07-08 | ||
| CN201180065989.9A CN103476928B (en) | 2010-12-09 | 2011-12-09 | For obtaining the method based on MGMT of high yield expression of recombinant proteins |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201180065989.9A Division CN103476928B (en) | 2010-12-09 | 2011-12-09 | For obtaining the method based on MGMT of high yield expression of recombinant proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107090441A true CN107090441A (en) | 2017-08-25 |
Family
ID=44279107
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201180065989.9A Active CN103476928B (en) | 2010-12-09 | 2011-12-09 | For obtaining the method based on MGMT of high yield expression of recombinant proteins |
| CN201610082061.6A Pending CN105821078A (en) | 2010-12-09 | 2011-12-09 | MGMT-Based method for obtaining high yield of recombinant protein expression |
| CN201710166781.5A Pending CN107090441A (en) | 2010-12-09 | 2011-12-09 | The method based on MGMT for obtaining high yield expression of recombinant proteins |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201180065989.9A Active CN103476928B (en) | 2010-12-09 | 2011-12-09 | For obtaining the method based on MGMT of high yield expression of recombinant proteins |
| CN201610082061.6A Pending CN105821078A (en) | 2010-12-09 | 2011-12-09 | MGMT-Based method for obtaining high yield of recombinant protein expression |
Country Status (8)
| Country | Link |
|---|---|
| US (3) | US9109219B2 (en) |
| EP (2) | EP2649178B8 (en) |
| JP (2) | JP5940554B2 (en) |
| CN (3) | CN103476928B (en) |
| BR (1) | BR112013014457A8 (en) |
| CA (1) | CA2819552C (en) |
| ES (1) | ES2627117T3 (en) |
| WO (1) | WO2012076715A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828738A (en) * | 2017-11-28 | 2018-03-23 | 新乡医学院 | A kind of dnmt rna deficiency Chinese hamster ovary celI system and preparation method and application |
| WO2020076174A1 (en) | 2018-10-09 | 2020-04-16 | Ibmc - Instituto De Biologia Molecular E Celular | Nucleic acid to activate gene expression and protein production |
| CN112359063A (en) * | 2020-11-04 | 2021-02-12 | 安徽环球基因科技有限公司 | Production method of novel crown recombinant RBD protein in insect cells |
| CN113039272A (en) * | 2018-08-14 | 2021-06-25 | 上海药明生物技术有限公司 | Transcription regulatory element and application thereof in enhancing expression of heterologous protein |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103476928B (en) | 2010-12-09 | 2017-03-29 | 巴斯德研究所 | For obtaining the method based on MGMT of high yield expression of recombinant proteins |
| EP2567968A1 (en) | 2011-09-09 | 2013-03-13 | Institut Pasteur | Polypeptides targeting glycosylated Muc2 proteins, methods of synthesis, their nucleic acids and uses therof |
| AU2012350229B2 (en) | 2011-12-09 | 2018-03-22 | Institut Pasteur | Multiplex immuno screening assay |
| AU2014354797C1 (en) * | 2013-11-29 | 2018-02-01 | Inovio Pharmaceuticals, Inc. | MERS-CoV vaccine |
| JP7012365B2 (en) * | 2016-02-25 | 2022-03-04 | ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア | New vaccine against Zika virus |
| CN105861753A (en) * | 2016-05-25 | 2016-08-17 | 深圳市第三人民医院 | Nested RT-PCR method, primers and reagent kit for detecting Zika virus |
| FR3052779B1 (en) * | 2016-06-17 | 2018-06-22 | Universite De Tours | NOVEL FUSION PROTEINS AND THEIR APPLICATION FOR THE PREPARATION OF VACCINES |
| CN106226529B (en) * | 2016-07-14 | 2018-03-06 | 四川农业大学 | The mark LDH of coenosis and the kit for diagnosing coenosis |
| CA3037486A1 (en) * | 2016-09-19 | 2018-03-22 | The Trustees Of The University Of Pennsylvania | Combination of novel vaccines against zika virus and dna antibody constructs for use against zika virus |
| US20200362313A1 (en) * | 2017-09-13 | 2020-11-19 | Biontech Rna Pharmaceuticals Gmbh | Method of enhancing rna expression in a cell |
| CN110531077B (en) * | 2018-05-25 | 2023-07-07 | 荣昌生物制药(烟台)股份有限公司 | Mesothelin immunohistochemical detection kit |
| KR102365464B1 (en) * | 2019-12-24 | 2022-02-22 | 강원대학교산학협력단 | Development of recombinant subunit Zika virus vaccine and preparing method thereof |
| WO2022020234A2 (en) * | 2020-07-20 | 2022-01-27 | Bio-Rad Laboratories, Inc. | Immunoassay for sars-cov-2 neutralizing antibodies and materials therefor |
| CN114908119A (en) * | 2021-02-09 | 2022-08-16 | 佛山汉腾生物科技有限公司 | Method for improving expression quantity of recombinant protein |
| CN115976104A (en) * | 2023-01-03 | 2023-04-18 | 中国食品药品检定研究院 | Method for purifying protein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005085470A1 (en) * | 2004-03-02 | 2005-09-15 | Epfl Ecole Polytechnique Federale De Lausanne | Specific substrates for o6- alkylguanine-dna alkyltransferase |
| WO2006114409A1 (en) * | 2005-04-27 | 2006-11-02 | Covalys Biosciences Ag | Pyrimidines reacting with o6-alkylguanine-dna alkyltransferase |
| CN101828113A (en) * | 2007-07-25 | 2010-09-08 | 德国弗劳恩霍夫协会债权安格万特学术研究所 | Self coupling recombinant antibody fusion proteins |
Family Cites Families (37)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2125047B (en) | 1982-08-09 | 1986-02-19 | Ciba Geigy Ag | Yeast hybrid vectors and their use for the production of polypeptides |
| EP0118551A1 (en) | 1982-09-15 | 1984-09-19 | Collaborative Research Inc. | Production of interferon in yeast by use of invertase promoter |
| US4758512A (en) | 1984-03-06 | 1988-07-19 | President And Fellows Of Harvard College | Hosts and methods for producing recombinant products in high yields |
| AU6543286A (en) | 1985-10-25 | 1987-05-19 | Zymogenetics Inc. | Method of using bar1 for secreting foreign proteins |
| US5681713A (en) | 1987-05-08 | 1997-10-28 | Smithkline Beecham Corporation | Expression of heterologous proteins in Drosophila cells |
| US5550043A (en) | 1987-05-08 | 1996-08-27 | Smithkline Beecham Corporation | Expression of heterologous proteins in Drosophila cells |
| US5879926A (en) | 1989-03-31 | 1999-03-09 | Transgene S.A. | Yeast strains for the production of mature heterologous proteins, especially hirudin |
| US5264618A (en) | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
| DK82893D0 (en) | 1993-07-08 | 1993-07-08 | Novo Nordisk As | PEPTIDE |
| FR2714830B1 (en) | 1994-01-10 | 1996-03-22 | Rhone Poulenc Rorer Sa | Composition containing nucleic acids, preparation and uses. |
| FR2715847B1 (en) | 1994-02-08 | 1996-04-12 | Rhone Poulenc Rorer Sa | Composition containing nucleic acids, preparation and uses. |
| FR2727679B1 (en) | 1994-12-05 | 1997-01-03 | Rhone Poulenc Rorer Sa | NEW TRANSFECTION AGENTS AND THEIR PHARMACEUTICAL APPLICATIONS |
| FR2730637B1 (en) | 1995-02-17 | 1997-03-28 | Rhone Poulenc Rorer Sa | PHARMACEUTICAL COMPOSITION CONTAINING NUCLEIC ACIDS, AND USES THEREOF |
| US5837520A (en) | 1995-03-07 | 1998-11-17 | Canji, Inc. | Method of purification of viral vectors |
| US5919682A (en) | 1995-08-24 | 1999-07-06 | Board Of Regents, University Of Texas System | Overproduction of neuronal nitric oxide synthase |
| AU3447097A (en) | 1996-07-01 | 1998-01-21 | Rhone-Poulenc Rorer S.A. | Method for producing recombinant adenovirus |
| CA2264482A1 (en) | 1996-09-06 | 1998-03-12 | The Trustees Of The University Of Pennsylvania | An inducible method for production of recombinant adeno-associated viruses utilizing t7 polymerase |
| WO1998017684A2 (en) * | 1996-10-25 | 1998-04-30 | Advanced Research & Technology Institute | Dna sequences encoding fusions of dna repair proteins and uses thereof |
| ATE348155T1 (en) | 1996-11-20 | 2007-01-15 | Introgen Therapeutics Inc | AN IMPROVED METHOD FOR PRODUCTION AND PURIFICATION OF ADENOVIRAL VECTORS |
| EP1679368B1 (en) | 1996-12-13 | 2010-08-11 | Schering Corporation | Methods for purifying viruses |
| FR2777909B1 (en) | 1998-04-24 | 2002-08-02 | Pasteur Institut | USE OF TRIPLEX-STRUCTURED DNA SEQUENCES FOR THE TRANSFER OF NUCLEOTID SEQUENCES IN CELLS, RECOMBINANT VECTORS CONTAINING THESE TRIPLEX SEQUENCES |
| JP3864610B2 (en) | 1998-05-21 | 2007-01-10 | 旭硝子株式会社 | Water-dispersed water / oil repellent composition and method for producing the same |
| DE69940785D1 (en) | 1998-11-20 | 2009-06-04 | Fuso Pharmaceutical Ind | PROTEIN EXPRESSION VECTOR AND USE OF THIS |
| KR100762033B1 (en) | 1998-12-31 | 2007-10-04 | 아방티 파르마 소시에테 아노님 | Method for separating viral particles |
| EP1155120B1 (en) | 1999-02-22 | 2006-07-05 | Transgene S.A. | Method for obtaining a purified viral preparation |
| ES2447115T3 (en) | 1999-10-11 | 2014-03-11 | Institut Pasteur | Vectors for the preparation of immunotherapeutic compositions |
| WO2001072783A2 (en) * | 2000-03-24 | 2001-10-04 | Genencor International, Inc. | Production of secreted proteins by recombinant eukaryotic cells |
| US8022172B2 (en) * | 2001-08-28 | 2011-09-20 | Allergan, Inc. | Luminescence resonance energy transfer (LRET) assays for clostridial toxin activity |
| US6682507B2 (en) | 2002-02-20 | 2004-01-27 | Doug's Kangaroo Pouch, Llc | User wearable device having sterile environment for connecting peritoneal dialysis tubes |
| NZ538986A (en) | 2002-10-03 | 2006-06-30 | Ecole Polytech | Substrates for O6-alkylguanine-DNA alkyltransferase |
| CA2501061A1 (en) * | 2002-10-03 | 2004-04-15 | Ecole Polytechnique Federale De Lausanne (Epfl) | Protein labelling with o6-alkylguanine-dna alkyltransferase |
| CN101595228A (en) * | 2005-07-21 | 2009-12-02 | 艾博特公司 | The method that comprises multi-gene expression and use polyprotein, precursor protein and the proteolysis of SORF construct |
| US20100196889A1 (en) * | 2006-11-13 | 2010-08-05 | Bankaitis-Davis Danute M | Gene Expression Profiling for Identification, Monitoring and Treatment of Colorectal Cancer |
| WO2008106551A2 (en) * | 2007-02-28 | 2008-09-04 | The Govt. Of The U.S.A. As Represented By The Secretary Of The Dept. Of Health & Human Serv. | Brachyury polypeptides and methods for use |
| FR2934684B1 (en) * | 2008-07-31 | 2012-11-16 | Cis Bio Int | METHOD OF DETECTING INTERNALIZATION OF MEMBRANE PROTEINS. |
| CN103476928B (en) * | 2010-12-09 | 2017-03-29 | 巴斯德研究所 | For obtaining the method based on MGMT of high yield expression of recombinant proteins |
| AU2012350229B2 (en) * | 2011-12-09 | 2018-03-22 | Institut Pasteur | Multiplex immuno screening assay |
-
2011
- 2011-12-09 CN CN201180065989.9A patent/CN103476928B/en active Active
- 2011-12-09 CA CA2819552A patent/CA2819552C/en active Active
- 2011-12-09 CN CN201610082061.6A patent/CN105821078A/en active Pending
- 2011-12-09 WO PCT/EP2011/072387 patent/WO2012076715A1/en active Application Filing
- 2011-12-09 JP JP2013542568A patent/JP5940554B2/en active Active
- 2011-12-09 ES ES11791602T patent/ES2627117T3/en active Active
- 2011-12-09 BR BR112013014457A patent/BR112013014457A8/en not_active Application Discontinuation
- 2011-12-09 EP EP11791602.3A patent/EP2649178B8/en active Active
- 2011-12-09 US US13/824,476 patent/US9109219B2/en active Active
- 2011-12-09 CN CN201710166781.5A patent/CN107090441A/en active Pending
- 2011-12-09 EP EP16207377.9A patent/EP3202897B1/en active Active
-
2015
- 2015-07-28 US US14/811,079 patent/US9546380B2/en active Active
-
2016
- 2016-02-29 JP JP2016038333A patent/JP6316856B2/en active Active
- 2016-12-13 US US15/377,554 patent/US10017769B2/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005085470A1 (en) * | 2004-03-02 | 2005-09-15 | Epfl Ecole Polytechnique Federale De Lausanne | Specific substrates for o6- alkylguanine-dna alkyltransferase |
| WO2006114409A1 (en) * | 2005-04-27 | 2006-11-02 | Covalys Biosciences Ag | Pyrimidines reacting with o6-alkylguanine-dna alkyltransferase |
| CN101828113A (en) * | 2007-07-25 | 2010-09-08 | 德国弗劳恩霍夫协会债权安格万特学术研究所 | Self coupling recombinant antibody fusion proteins |
Non-Patent Citations (1)
| Title |
|---|
| VOELTER,V.: "methylated-DNA--protein-cysteine methyltransferase [Homo sapiens]，NP_002403.2", 《NCBI GENBANK》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828738A (en) * | 2017-11-28 | 2018-03-23 | 新乡医学院 | A kind of dnmt rna deficiency Chinese hamster ovary celI system and preparation method and application |
| CN113039272A (en) * | 2018-08-14 | 2021-06-25 | 上海药明生物技术有限公司 | Transcription regulatory element and application thereof in enhancing expression of heterologous protein |
| CN113039272B (en) * | 2018-08-14 | 2023-05-30 | 上海药明生物技术有限公司 | Transcriptional regulatory elements and their use in enhancing expression of heterologous proteins |
| WO2020076174A1 (en) | 2018-10-09 | 2020-04-16 | Ibmc - Instituto De Biologia Molecular E Celular | Nucleic acid to activate gene expression and protein production |
| CN112359063A (en) * | 2020-11-04 | 2021-02-12 | 安徽环球基因科技有限公司 | Production method of novel crown recombinant RBD protein in insect cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103476928A (en) | 2013-12-25 |
| CN105821078A (en) | 2016-08-03 |
| JP2014502163A (en) | 2014-01-30 |
| CN103476928B (en) | 2017-03-29 |
| CA2819552C (en) | 2023-09-26 |
| CA2819552A1 (en) | 2012-06-14 |
| EP2649178B8 (en) | 2017-08-30 |
| WO2012076715A1 (en) | 2012-06-14 |
| US20170088843A1 (en) | 2017-03-30 |
| US20160017367A1 (en) | 2016-01-21 |
| EP3202897B1 (en) | 2020-04-15 |
| JP6316856B2 (en) | 2018-04-25 |
| EP2649178B1 (en) | 2017-03-15 |
| ES2627117T3 (en) | 2017-07-26 |
| EP3202897A1 (en) | 2017-08-09 |
| EP2649178A1 (en) | 2013-10-16 |
| US9546380B2 (en) | 2017-01-17 |
| US10017769B2 (en) | 2018-07-10 |
| US20130309747A1 (en) | 2013-11-21 |
| BR112013014457A8 (en) | 2017-12-19 |
| JP2016154543A (en) | 2016-09-01 |
| BR112013014457A2 (en) | 2016-09-27 |
| US9109219B2 (en) | 2015-08-18 |
| JP5940554B2 (en) | 2016-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107090441A (en) | The method based on MGMT for obtaining high yield expression of recombinant proteins | |
| KR102370675B1 (en) | Improved methods for modification of target nucleic acids | |
| AU2014273089B2 (en) | A LAGLIDADG homing endonuclease cleaving the C-C Chemokine Receptor Type-5 (CCR5) gene and uses thereof | |
| CN101595220B (en) | Attenuated recombinant newcastle disease virus and vaccine containing the same | |
| CN108642053A (en) | The sgRNA of CRISPR-Cas9 targeting knock out people colon-cancer cell PPP1R1C genes and its specificity | |
| CN108588071A (en) | The sgRNA of CRISPR-Cas9 targeting knock out people colon-cancer cell CNR1 genes and its specificity | |
| CN107893075A (en) | CRISPR Cas9 targeting knock out people colon-cancer cell RITA genes and its specific sgRNA | |
| KR101742346B1 (en) | Lysosomal storage disease enzyme | |
| CA2320957A1 (en) | Alteration of amino acid compositions in seeds | |
| CN107868780B (en) | Method for realizing site-directed mutation on circular DNA molecule larger than 10kb | |
| CN109475619A (en) | The gene therapy of neuronal waxy lipofuscinosis | |
| CN114058604A (en) | Fusion protein and application thereof in base editing | |
| KR20200015900A (en) | Self-inactivating virus vector | |
| KR100862049B1 (en) | Attenuated recombinant newcastle disease virus | |
| JP2022542828A (en) | Recombinant modified adeno-associated virus helper vectors and their use to improve packaging efficiency of recombinant modified adeno-associated virus | |
| CN105176936B (en) | Replicate the subclone and preparation method and application of the Semliki forest virus of tolerance type | |
| CN116135974A (en) | Recombinant glycosylase base editing system and application thereof | |
| CN113061626B (en) | Method for knocking out zebra fish genes in tissue-specific manner and application | |
| CN111534542A (en) | PiggyBac transposon system mediated eukaryotic transgenic cell line and construction method thereof | |
| KR102315601B1 (en) | Recombinant vector and method for producing recombinant fibroblast growth factor 19 using the same | |
| KR20200132958A (en) | New EHV with UL18 and/or UL8 inactivated | |
| KR20080030378A (en) | System of biomolecular fluorescence complementation containing a cell inserted a part of fluorescence protein gene in chromosome and methods of biomolecular fluorescence complementation by using it | |
| KR20230159050A (en) | Whole Cell Biocatalyst Expressing Plastic Hydrolase and Carbohydrate-Binding Module On the Surface Thereof | |
| Kang | Mapping the Interactions of the Multi-Subunit General Transcription Factor IIIB (TFIIIB) with U6 Promoter DNA by Site-Specific Protein-DNA Photo-Cross-Linking | |
| CN112322655B (en) | Base editing system free from restriction of gene sequence, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170825 |
|
| WD01 | Invention patent application deemed withdrawn after publication |